JP5829245B2 - Albumin fusion protein - Google Patents

Description

(Background of the Invention)
The present invention relates generally to therapeutic proteins fused to albumin or albumin fragments or variants, including but not limited to at least one polypeptide, antibody, peptide, or fragments and variants thereof. The invention encompasses polynucleotides encoding therapeutic albumin fusion proteins, therapeutic albumin fusion proteins, compositions, pharmaceutical compositions, formulations and kits. Host cells transformed with a polynucleotide encoding a therapeutic albumin fusion protein are also included in the invention, and these polynucleotides and / or host cells are used to make the albumin fusion proteins of the invention. Methods are likewise included in the present invention.

  Human serum albumin (HSA, or HA), which in its mature form is a 585 amino acid protein (as shown in FIG. 1 (SEQ ID NO: 1038)), is responsible for an important part of the osmotic pressure of serum, and is composed of endogenous ligands and It also functions as a carrier for exogenous ligands. Currently, HA for clinical use is produced by extraction from human blood. The production of recombinant HA (rHA) in microorganisms is disclosed in EP330451 and EP361991.

  Therapeutic proteins (eg, interferon and growth hormone) in their native state or when produced recombinantly are typically unstable molecules that exhibit a short shelf life, especially formulated in aqueous solutions. In some cases, it is an unstable molecule that exhibits a short shelf life. The instability of these molecules when formulated for administration is that many of the molecules must be lyophilized or refrigerated at all times during storage, thereby transporting and / or storing the molecules. Dominate what will be difficult to do. Storage problems are particularly acute when pharmaceutical formulations must be stored and dispensed outside the hospital environment.

  Few practical solutions to the storage problem of unstable protein molecules have been proposed. Thus, there is a need for a stable, long lasting formulation of protein therapeutic molecules that is easily dispensed, preferably with a simple formulation that requires minimal post-storage operations.

  The invention encompasses albumin fusion proteins comprising therapeutic proteins (eg, polypeptides, antibodies, peptides, or fragments or variants thereof) fused to albumin or a fragment (part) or variant of albumin. . The invention includes or includes nucleic acid molecules that encode therapeutic proteins (eg, polypeptides, antibodies, peptides, or fragments or variants thereof) fused to albumin or a fragment (part) or variant of albumin. A polynucleotide consisting of The present invention is sufficient to prolong the shelf life of a therapeutic protein and / or stabilize its activity in vitro or in vivo in a therapeutic protein and / or in solution (or in a pharmaceutical composition), A polynucleotide comprising or consisting of a nucleic acid molecule encoding a therapeutic protein (eg, polypeptide, antibody, peptide, or fragment or variant thereof) fused to albumin or a fragment (part) or variant of albumin Also includes. Albumin fusion proteins encoded by the polynucleotides of the present invention are also encompassed by the present invention, using host cells transformed with the polynucleotides of the present invention, and using these polynucleotides and / or host cells of the present invention. Also encompassed by the present invention are methods of making the albumin fusion proteins of the present invention.

  In preferred aspects of the invention, albumin fusion proteins include, but are not limited to, those encoded by the polynucleotides listed in Table 2.

  The invention also includes a pharmaceutical formulation comprising an albumin fusion protein of the invention and a pharmaceutically acceptable excipient or carrier. Such a formulation may be present in a kit or container. Such a kit or container can be packaged with instructions for an extended shelf life of the therapeutic protein. Such a formulation can be used in a method of treating, preventing, ameliorating or diagnosing a disease or disease sign in a patient, preferably a mammal, most preferably a human, which method is used to treat the pharmaceutical formulation to the patient. The step of administering to.

  In other embodiments, the invention includes methods for preventing, treating, or ameliorating a disease or injury. In a preferred embodiment, the invention encompasses a method of treating a disease or disorder listed in the “Preferred indication Y” column of Table 1, wherein such method is desired for such treatment, prevention or amelioration. In an amount effective to treat, prevent or ameliorate the disease or disorder in the patient, the “Therapeutic Protein X” column of Table 1 (the disease or disorder being treated is the “Preferred indication Y” column of Table 1). Administering an albumin fusion protein of the invention comprising the disclosed therapeutic protein or a portion corresponding to the therapeutic protein (fragments or variants thereof).

  In one embodiment, the albumin fusion protein described in Table 1 or 2 has an extended shelf life.

  In a second embodiment, the albumin fusion protein described in Table 1 or 2 is more stable than the corresponding non-fusion therapeutic molecule described in Table 1.

  The invention further expresses an albumin fusion protein of the invention, preferably modified to include the nucleic acid molecules of the invention (including but not limited to the polynucleotides set forth in Tables 1 and 2). Including transgenic organisms modified.

FIG. 1A shows the amino acid sequence of the mature form of human albumin (SEQ ID NO: 1038) and the polynucleotide encoding it (SEQ ID NO: 1037). FIG. 1B shows the amino acid sequence of the mature form of human albumin (SEQ ID NO: 1038) and the polynucleotide encoding it (SEQ ID NO: 1037). FIG. 1C shows the amino acid sequence of the mature form of human albumin (SEQ ID NO: 1038) and the polynucleotide encoding it (SEQ ID NO: 1037). FIG. 1D shows the amino acid sequence of the mature form of human albumin (SEQ ID NO: 1038) and the polynucleotide encoding it (SEQ ID NO: 1037). FIG. 2 shows the restriction map of the pPPC0005 cloning vector (ATCC accession number PTA-3278). FIG. 3 shows pSAC35 yeast S. pneumoniae. 1 shows a restriction map of a cerevisiae expression vector (Sleep et al., Biotechnology 8:42 (1990)). FIG. 4 shows the EPO albumin fusion protein encoded by the DNA contained in the construct ID numbers (hereinafter CID) 1966 and 1981, and various dilutions of recombinant human EPO, for TF-1 cell growth. (See Examples 8 and 9). Cells were washed 3 times to remove GM-CSF and plated at 10,000 cells / well for 72 hours in the presence of CID1966 protein or a 3-fold dilution of CID1981 protein. The concentration used was calculated based on the weight of Epo alone, not HSA + Epo. Recombinant human Epo (rhEpo) was used as a positive control and serially diluted 3-fold from 100 ng / ml to 0.01 ng / ml. Cells were exposed to 0.5 mCi / well 3H-thymidine for an additional 18 hours. (White square) rhEpo; (reverse black triangle) HSA-Epo 1981; (black circle) Epo-HSA 1996. FIG. 5 is a dose response analysis of various doses of recombinant human EPO and EPO albumin fusion protein encoded by DNA contained in CID 1966 and CID 1981 relative to the% change in hematocrit from day 0 and day 7. The effect is shown (see Examples 8 and 9). 48-week-old female DBA / 2NHsd mice were divided into 12 groups of 4 animals each. Recombinant human EPO (rhEpo) was injected subcutaneously at days 0, 2, 4 and 6 at 0.5, 1.5, 4.5 and 12 μg / kg. Epo albumin fusion proteins made from constructs CID 1966 and CID 1981 were injected subcutaneously on days 0, 2, 4 and 6 at 2, 6, 18 and 54 μg / kg. Higher doses of Epo albumin fusion protein allow an approximately equimolar comparison with recombinant human Epo (note that the weight of the fusion is approximately 4.35 times the weight of non-glycosylated Epo) ). On days 0 and 7 of the experiment, blood was drawn from the animals via the tail vein and the hematocrit was determined by centrifugation. (Black square) rhEpo; (white circle) CID 1981; (reverse black triangle) CID 1966. FIG. 6A shows the effect of various subcutaneous administrations of Epo albumin fusion protein encoded by DNA contained in CID1966 and CID1997, respectively, on the% change in hematocrit from day 0 to day 8 (Examples 8 and 10). checking). *, P <0.005, compared to rhEpo as determined by Mann-Whitney non-parametric analysis (n = 6). FIG. 6B shows the effect of subcutaneous administration of Epo albumin fusion protein encoded by DNA contained in CID 1997 and CID 1966 on the% change in hematocrit from day 0 to day 14 (see Examples 8 and 10). about). *, P <0.005, as determined by Mann-Whitney non-parametric analysis, compared to rhEpo (n = 6); **, p <0.05, as determined by Mann-Whitney non-parametric analysis , RhEpo (n = 6). FIG. 7 shows the effect of various diluted albumin fusion proteins encoded by DNA contained in CID 1981 and CID 1997, respectively, on TF-1 cell proliferation (see Examples 9 and 10). Cells were washed 3 times to remove GM-CSF and plated at 10,000 cells / well for 72 hours in the presence of a 3-fold dilution of Epo albumin fusion protein encoded by CID1981 or CID1997. . An equimolar amount of rhEpo was used as a positive control (the weight of the non-glycosylated Epo is 20 kd, whereas the Epo albumin fusion protein is 87 kd, so about 1.35 times less protein was added. did). Cells were exposed to 0.5 μCi / well 3H-thymidine for an additional 24 hours. (Black square) rhEpo standard; (black triangle) CID 1981 (CHO); (white circle) CID 1997 (NSO). FIG. 8 shows the effect of different doses of recombinant human EPO (rhEpo) and EPO albumin fusion protein encoded by DNA contained in construct 1997 (CID 1997) on the% change in hematocrit from day 0 to day 8. (See Example 10). (Black triangle) = rhEpo; (white square) = CID1997. FIG. 9 shows the effect of various dilutions of IL2 albumin fusion protein encoded by DNA contained in CID 1812 on CTLL-2 proliferation (see Example 15). 1 × 10 ⁴ cells / well were seeded in 96-well plates in a final volume of 200 μl complete medium containing the indicated amount of IL2 albumin fusion protein (CID1812). All samples were done in triplicate. The cells were incubated at 37 ° C. for 40 minutes, then 20 μl Alamar Blue was added and the cells were incubated for 8 hours. An absorbance of 530/590 was used as a measure of proliferation. EC50 = 0.386 ± 0.021. (White triangle) = CID1812. FIG. 10 shows the effect of IL2 albumin fusion protein encoded by the DNA contained in CID 1812 on day 21 RENCA tumor growth (see Example 15). BALB / c mice (n = 10) were injected SC with 10 ⁵ RENCA cells (mid-flank). Ten days later, a daily injection (QD) of rIL2 (0.9 mg / kg), IL2 albumin fusion protein (CID1812 protein; 0.6 mg / kg), or PBS (placebo), or CID1812 protein (0.6 mg Mice were given every other day injection (QOD). Tumor volume was determined on day 21 after RENCA inoculation. Data are shown by scatter analysis (each dot represents one animal). The average value for each group is indicated by a horizontal line. *, P = 0.0035, between placebo control and CID1812 protein. Numbers in parentheses indicate the number of surviving mice relative to the total number of mice per group. (White circle) = placebo; (black circle) = IL2; (white triangle) = CID1812 protein (QD); (white square) = CID1812 protein (QOD). FIG. 11 shows the effect of various dilutions of GCSF albumin fusion protein encoded by DNA contained in CID1642 and CDI1643 on NFS-60 cell proliferation (see Examples 19 and 20). (Black square) = CID1642; (black triangle) = CID1643; (white circle) = HSA. FIG. 12 shows the effect of recombinant human GCSF (Neupogen) and GCSF albumin fusion protein on total white blood cell count (see Example 19). Total WBC on each day (10 ³ cells / μl) is shown as group mean ± SEM. The GCSF albumin fusion protein was injected sc at 4 × every 4 days (Q4D), either at 25 μg / kg or 100 μg / kg, or twice every 7 days (Q7D) at 100 μg / kg. Day 8 and day 9 data for GCSF albumin fusion protein 100 μg / kg Q7 are shown as day 9 and day 10, respectively, to facilitate comparison with other groups. Controls were saline vehicle injected 4 sc every 4 days (vehicle Q4D) or Neupogen (Neupogen 5 μg / kg QD) administered 14 sc once daily. The treatment period is considered to be 1 to 14 days and the recovery period is considered to be 15 to 28 days. FIG. 13 shows the effect of various dilutions of IFNb albumin fusion protein encoded by DNA contained in CID2011 and CID2053 on SEAP activity in ISRE-SEAP / 293F reporter cells (see Example 25). The protein was serially diluted from 5e-7 to le-14 g / ml in DMEM / 10% FBS and used to treat ISRE-SEAP / 293F reporter cells. After 24 hours, the supernatant was removed from the reporter cells and assayed for SEAP activity. The IFNb albumin fusion protein was purified from three stable clones: 293F / # 2011, CHO / # 2011 and NSO / # 2053. Avonex, a mammal-derived IFNb, was produced by Biogen and was reported to have a specific activity of 2.0e5 IU / μg. FIG. 14 illustrates the constant level of insulin mRNA in INS-1 (832/13) cells after treatment with GLP-1 or GLP-1 albumin fusion protein encoded by construct ID3070 (CID3070 protein). Both GLP-1 and CID3070 proteins stimulate transcription of the insulin gene in INS-1 cells. The first bar (black) shows untreated cells. Bars 2-4 (white) represent cells treated with the indicated concentrations of GLP-1. Bars 5-7 (grey) represent cells treated with the indicated concentrations of CID3070 protein. FIG. 15 compares the antiproliferative activity of IFN albumin fusion protein encoded by CID3165 (CID3165 protein) and recombinant IFNa (fINFα) against Hs294T melanoma cells. Cells were cultured with various concentrations of either CID 3165 protein or rIFNa, and proliferation was measured by BrdU incorporation after 3 days of culture. CID 3165 protein caused measurable inhibition of cell proliferation at concentrations above 10 ng / ml, and 50% inhibition was achieved at about 200 ng / ml. (Black square) = CID3165 protein, (black diamond) = rIFNa. FIG. 16 shows the effect of various contracts of IFNa albumin fusion protein on SEAP activity in ISRE-SEAP / 293F reporter cells. One preparation of IFNa fused to albumin upstream (black diamond) was tested, and two different preparations of IFNa fused to albumin downstream (black triangle) and (black square) were also tested. FIG. 17 shows the effect of time and dose of IFNa albumin fusion protein encoded by the DNA contained in construct 2249 (CID2249 protein) on mRNA levels of OAS (p41) in treated monkeys (see Example 31). ). Per time point: first bar = vehicle control, second bar = 30 μg / kg CID2249 protein day 1 iv, third bar = 30 μg / kg CID2249 protein day 1 sc, fourth bar = 300 μg / kg CID2249 protein Day 1 sc, 5th bar = 40 μg / kg Recombinant IFNa Day 1, Day 3, and Day 5 sc. FIG. 18 shows the effect of various dilutions of insulin albumin fusion protein encoded by DNA contained in constructs 2250 and 2276 on glucose uptake in 3T3-Ll adipocytes (see Examples 33 and 35). . FIG. 19 shows the effect of various GCSF albumin fusion proteins (including those encoded by CID # 1643 and CID # 2702 (see L-171, Example 114)) on NFS cell proliferation. A horizontal broken line indicates a minimum detection level.

(Detailed description of the invention)
(Definition)
The following definitions are provided to facilitate understanding of certain terms used throughout this specification.

  As used herein, “polynucleotide” refers to at least one molecule of albumin (or fragment thereof) linked in-frame to at least one therapeutic protein X (or fragment or variant thereof). Or a nucleic acid molecule having a nucleotide sequence that encodes a fusion protein consisting thereof; an amino acid sequence of SEQ ID NO: Y (described in column 6 of Table 2) or a fragment or variant thereof Alternatively, a nucleic acid molecule having a nucleotide sequence encoding a fusion protein comprising these; a nucleic acid molecule comprising the sequence shown in or consisting of SEQ ID NO: X; a fusion protein comprising or consisting of the amino acid sequence of SEQ ID NO: Z Code A nucleic acid molecule having a tide sequence; a nucleic acid molecule having a nucleotide sequence encoding an albumin fusion protein of the invention produced as described in Table 2 or the Examples; a nucleotide sequence encoding a therapeutic albumin fusion protein of the invention A nucleic acid molecule having a nucleotide sequence contained in an albumin fusion construct described in Table 2 or an albumin fusion construct deposited with the ATCC (described in Table 3). A nucleic acid molecule.

  As used herein, an “albumin fusion construct” is at least one linked in-frame to at least one polynucleotide encoding at least one molecule of therapeutic protein (or fragment or variant thereof). A nucleic acid molecule comprising or consisting of a polynucleotide encoding the molecule albumin (or a fragment or variant thereof); at least one molecule of a therapeutic protein (or its) produced as described in Table 2 or in the Examples A nucleic acid molecule comprising or consisting of at least one albumin (or fragment or variant thereof) that is fused in-frame to at least one polynucleotide encoding (fragment or variant); For example, the following elements: (1) a functional self-replicating vector (shuttle vector, expression vector, integration vector, and / or replication system), (2) a region for initiation of transcription (eg, a regulatable promoter or Therapeutic of at least one molecule further comprising one or more of an inducible promoter, a promoter region such as a constitutive promoter), (3) a region for termination of transcription, (4) a leader sequence, and (5) a selectable marker. A polynucleotide encoding or consisting of at least one molecule of albumin (or a fragment or variant thereof) linked in frame to at least one polynucleotide encoding a protein for use (or fragment or variant thereof) Nucleic acid. A polynucleotide encoding a therapeutic protein and an albumin protein can be referred to as a “part,” “region,” or “part,” respectively, of an albumin fusion construct once it is part of an albumin fusion construct.

  The present invention relates generally to polynucleotides encoding albumin fusion proteins; albumin fusion proteins; albumin fusion proteins or methods of treating or preventing or ameliorating a disease or disorder using polynucleotides encoding albumin fusion proteins. As used herein, an “albumin fusion protein” is a fusion of at least one molecule of albumin (or a fragment or variant thereof) to at least one molecule of a therapeutic protein (or fragment or variant thereof). Refers to the protein formed by The albumin fusion proteins of the invention include at least a fragment or variant of a therapeutic protein and at least a fragment or variant of human serum albumin that are linked together by genetic fusion (ie, an albumin fusion protein is a therapeutic A polynucleotide that encodes all or part of the protein of interest is generated by translation of a nucleic acid linked in-frame to a polynucleotide that encodes all or part of albumin). Once the therapeutic protein and albumin protein are part of the albumin fusion protein, each “portion”, “region” or “moiety” (eg, “therapeutic protein portion” of the albumin fusion protein). Or “albumin protein portion”). In a highly preferred embodiment, the albumin fusion protein of the invention comprises at least one molecule of therapeutic protein X or a fragment or variant thereof (including but not limited to a mature form of therapeutic protein X) and at least one molecule. Or a fragment or variant thereof, including but not limited to mature forms of albumin.

In a further preferred embodiment, the albumin fusion protein of the invention is processed by the host cell and secreted into the surrounding culture medium. Processing of the nascent albumin fusion protein that occurs in the secretory pathway of the host used for expression involves signal peptide cleavage; disulfide bond formation; proper folding; carbohydrate addition and processing (eg, N-linked glycosylation and O-linkage). Type glycosylation); specific protein cleavage; and assembly into multimeric proteins.
The albumin fusion protein of the present invention is preferably in a processed form. In a most preferred embodiment, “processed form of albumin fusion protein” refers to an albumin fusion protein product that has undergone N-terminal signal peptide cleavage (also referred to herein as “mature albumin fusion protein”).

  In some examples, a representative clone containing an albumin fusion construct of the invention was deposited with the American Type Culture Collection (referred to herein as “ATCC®”). Furthermore, it is possible to recover a predetermined albumin fusion construct from the deposit by techniques known in the art and described herein. ATCC (R) is located at 10801 University Boulevard, Manassas, Virginia 20110-2209, USA. The ATCC deposit was made in accordance with the provisions of the Budapest Treaty on the international recognition of the deposit of microorganisms for patent procedure purposes.

In one embodiment, the present invention provides a polynucleotide encoding an albumin fusion protein comprising or consisting of a therapeutic protein and serum albumin protein. In a further embodiment, the present invention provides an albumin fusion protein comprising or consisting of a therapeutic protein and serum albumin protein.
In a preferred embodiment, the present invention provides an albumin fusion protein comprising or consisting of a therapeutic protein encoded by a polynucleotide set forth in Table 2 and a serum albumin protein. In a further preferred embodiment, the present invention provides a polynucleotide encoding an albumin fusion protein whose sequence is shown in Table 2 as SEQ ID NO: Y. In other embodiments, the present invention provides biologically active and / or therapeutically active fragments of therapeutic proteins, and albumin fusion proteins comprising or consisting of serum albumin proteins. In other embodiments, the present invention provides biologically active and / or therapeutically active variants of therapeutic proteins, and albumin fusion proteins comprising or consisting of serum albumin proteins. In a preferred embodiment, the serum albumin protein component of the albumin fusion protein is the mature form of serum albumin. The present invention further includes polynucleotides encoding these albumin fusion proteins.

  In further embodiments, the present invention provides albumin fusion proteins comprising or consisting of therapeutic proteins and biologically active and / or therapeutically active fragments of serum albumin. In further embodiments, the present invention provides albumin fusion proteins comprising or consisting of therapeutic proteins and biologically active and / or therapeutically active variants of serum albumin. In preferred embodiments, the therapeutic protein portion of the albumin fusion protein is the mature form of the therapeutic protein. In a further preferred embodiment, the therapeutic protein portion of the albumin fusion protein is the extracellular soluble domain of the therapeutic protein. In an alternative embodiment, the therapeutic protein portion of the albumin fusion protein is the active form of the therapeutic protein. The present invention further includes polynucleotides encoding these albumin fusion proteins.

  In further embodiments, the present invention provides biologically active and / or therapeutically active fragments or variants of therapeutic proteins, and biologically active and / or therapeutically active of serum albumin. Albumin fusion proteins comprising or consisting of fragments or variants are provided. In a preferred embodiment, the present invention provides an albumin fusion protein comprising or consisting of a mature portion of a therapeutic protein and a mature form of serum albumin. The present invention further includes polynucleotides encoding these albumin fusion proteins.

(Therapeutic protein)
As mentioned above, the polynucleotide of the invention encodes a protein comprising or consisting of at least a fragment or variant of a therapeutic protein and at least a fragment or variant of human serum albumin, which are preferably linked to each other, preferably They are linked by genetic fusion.

  Further embodiments encode a protein comprising or consisting of at least a fragment or variant of a therapeutic protein and at least a fragment or variant of human serum albumin, which are linked to each other by chemical conjugation. .

  As used herein, “therapeutic protein” refers to a protein, polypeptide, antibody, peptide or fragment or variant thereof having one or more therapeutic and / or biological activities. Therapeutic proteins encompassed by the present invention include, but are not limited to, proteins, polypeptides, peptides, antibodies, and biologics (the terms peptides, proteins, and polypeptides are defined herein). Used interchangeably in the book). The term “therapeutic protein” encompasses antibodies and fragments or variants thereof. Thus, the protein of the invention may comprise at least a fragment or variant of a therapeutic protein and / or at least a fragment or variant of an antibody. Furthermore, the term “therapeutic protein” may refer to an endogenous or naturally occurring correlator of the therapeutic protein.

  Polypeptides that exhibit “therapeutic activity” or “therapeutically active” proteins, such as the therapeutic proteins described herein or one or more therapeutic proteins otherwise known in the art. Means a polypeptide possessing one or more known biological and / or therapeutic activities associated with a therapeutic protein. As a non-limiting example, a “therapeutic protein” is a protein useful for treating, preventing, or ameliorating a disease, condition, or disorder. By way of non-limiting example, a “therapeutic protein” is a protein that specifically binds to a specific cell type (normal (eg, lymphocyte) or abnormal (eg, cancer cell)), and therefore the cell type. It can be used to specifically target a compound (drug or cytotoxic agent).

  For example, a non-exhaustive list of “therapeutic proteins” portions that can be included by the albumin fusion proteins of the invention includes, but is not limited to: erythropoietin (EPO), IL-2, G-CSF, insulin, Calcitonin, growth hormone, IFN-α, IFN-β, PTH, TR6 (International Publication No. WO 98/30694), BLyS, BLyS single chain antibody, Resistin, growth hormone releasing factor, VEGF-2, KGF-2, D-SLAM , KDI, and TR2, GLP-1, Extendin 4 and GM-CSF.

The interferon hybrid can also be fused to the amino terminus or carboxy terminus of albumin to form an interferon hybrid albumin fusion protein.
Interferon hybrid albumin fusion proteins can enhance interferon activity (eg, antiviral response, modulation of cell proliferation and immune response) or suppress interferon activity (Lebleu et al., PNAS USA, 73: 3107-). 3111 (1976); Gresser et al., Nature, 251: 543-545 (1974); and Johnson, Texas Reports Biol Med, 35: 357-369 (1977)). Each interferon hybrid albumin fusion protein can be used to treat, prevent or ameliorate viral infection (eg, hepatitis (eg, HCV); or HIV), multiple sclerosis, or cancer.

  In one embodiment, the interferon hybrid portion of the interferon hybrid albumin fusion protein comprises an interferon α-interferon α hybrid (referred to herein as an α-α hybrid). For example, the α-α hybrid portion of the interferon hybrid albumin fusion protein consists of or includes interferon α A fused to interferon α D. In a further embodiment, the A / D hybrid is fused at a BgE restriction site common to interferon αD. Here, the N-terminal portion of the A / D hybrid corresponds to amino acids 1-62 of interferon α A, and the C-terminal portion corresponds to amino acids 64-166 of interferon α D. For example, this A / D hybrid comprises the following amino acid sequence:

ここでＸ_１は、ＲまたはＫであり、Ｘ_２はＡまたはＶであり（例えば、構築物＃２８７５を参照のこと）。さらなる実施形態において、このＡ／Ｄハイブリッドは、一般的なＰｖｕＩＩＩ制限部位において融合される。ここでこのＡ／ＤハイブリッドのＮ末端部分は、インターフェロンα Ａのアミノ酸１−９１に対応し、Ｃ末端部分は、インターフェロンα Ｄのアミノ酸９３−１６６に対応する。例えば、このＡ／Ｄハイブリッドは、以下のアミノ酸配列を含む：

Where X ₁ is R or K and X ₂ is A or V (see, eg, Construct # 2875). In a further embodiment, the A / D hybrid is fused at a common PvuIII restriction site. Here, the N-terminal portion of the A / D hybrid corresponds to amino acids 1-91 of interferon α A, and the C-terminal portion corresponds to amino acids 93-166 of interferon α D. For example, this A / D hybrid comprises the following amino acid sequence:

ここでＸ_１は、ＲまたはＫであり、第２のＸ_２は、ＡまたはＶである（例えば、構築物ＩＤ＃２８７２を参照のこと）。これらのハイブリッドは、米国特許第４，４１４，５１０号（これは、その全体が本明細書中に参考として援用される）にさらに記載される。

Where X ₁ is R or K, and the second X ₂ is A or V (see, eg, Construct ID # 2872). These hybrids are further described in US Pat. No. 4,414,510, which is hereby incorporated by reference in its entirety.

  In a further embodiment, the alpha-alpha hybrid portion of the interferon hybrid albumin fusion protein consists of or comprises interferon alpha A fused to interferon alpha F. In a further embodiment, the A / F hybrid is fused to a general PvuIII restriction site, wherein the N-terminal portion of the A / F hybrid corresponds to amino acids 1-91 of interferon α A and the C-terminal portion is , Corresponding to amino acids 93-166 of interferon α F. For example, this A / F hybrid comprises the following amino acid sequence:

ここでＸはＲまたはＫのいずれかである（例えば、構築物ＩＤ＃２８７４）。これらのハイブリッドは、米国特許第４，４１４，５１０号（これは、その全体が本明細書中に参考として援用される）にさらに記載される。さらなる実施形態において、このインターフェロンハイブリッドアルブミン融合タンパク質のα−αハイブリッド部分は、インターフェロンα Ｂに融合されたインターフェロンα Ａからなるかあるいはこれらを含む。代替的実施形態において、このＡ／Ｂハイブリッドは、一般的なＰｖｕＩＩＩ制限部位において融合され、ここでこのＡ／ＢハイブリッドのＮ末端部分は、インターフェロンα Ａのアミノ酸１−９１に対応し、Ｃ末端部分は、インターフェロンα Ｂのアミノ酸９３−１６６に対応する。例えば、このＡ／Ｂハイブリッドは、以下のアミノ酸配列を含む：

Here, X is either R or K (for example, construct ID # 2874). These hybrids are further described in US Pat. No. 4,414,510, which is hereby incorporated by reference in its entirety. In further embodiments, the α-α hybrid portion of the interferon hybrid albumin fusion protein consists of or comprises interferon α A fused to interferon α B. In an alternative embodiment, the A / B hybrid is fused at a general PvuIII restriction site, where the N-terminal portion of the A / B hybrid corresponds to amino acids 1-91 of interferon α A and is C-terminal. The portion corresponds to amino acids 93-166 of interferon αB. For example, this A / B hybrid comprises the following amino acid sequence:

ここでＸ_１はＲまたはＫであり、Ｘ_２からＸ_５は、ＳＣＶＭまたはＶＬＣＤである（例えば、構築物ＩＤ＃２８７３を参照のこと）。これらのハイブリッドは、米国特許第４，４１４，５１０号（これは、その全体が本明細書中に参考として援用される）にさらに記載される。

Where X ₁ is R or K and X ₂ to X ₅ are SCVM or VLCD (see, eg, Construct ID # 2873). These hybrids are further described in US Pat. No. 4,414,510, which is hereby incorporated by reference in its entirety.

  In another embodiment, the interferon hybrid portion of the interferon hybrid albumin fusion protein comprises an interferon β-interferon α hybrid (referred to herein as a β-α hybrid). For example, the β-α hybrid portion of the interferon hybrid albumin fusion protein consists of or includes interferon β-1 fused to interferon αD (also referred to as interferon α-1). In a further embodiment, the β-1 / α D hybrid is fused, wherein the N-terminal portion corresponds to amino acids 1-73 of interferon β-1 and the C-terminal portion is amino acids 74-167 of interferon α D. Corresponding to For example, this β-1 / α D hybrid comprises the following amino acid sequence:

ここでＸは、ＡまたはＶである。これらのハイブリッドは、米国特許第４，７５８，４２８号（これは、その全体が本明細書中に参考として援用される）においてさらに記載される。

Here, X is A or V. These hybrids are further described in US Pat. No. 4,758,428, which is hereby incorporated by reference in its entirety.

In another embodiment, the interferon hybrid portion of the interferon hybrid albumin fusion protein comprises an interferon α-interferon β hybrid (referred to herein as an α-β hybrid). For example, the α-β hybrid portion of the interferon hybrid albumin fusion protein consists of or includes interferon αD (also referred to as interferon α-1) fused to interferon β-1. In a further embodiment, the αD / β-1 hybrid is fused, wherein the N-terminal portion corresponds to amino acids 1-73 of interferon αD and the C-terminal portion is amino acids 74-166 of interferon β-1. Correspond.
For example, the αD / β-1 hybrid has the following amino acid sequence:

これらのハイブリッドは、米国特許第４，７５８，４２８号（これは、その全体が本明細書中に参考として援用される）にさらに記載される。

These hybrids are further described in US Pat. No. 4,758,428, which is hereby incorporated by reference in its entirety.

  In further embodiments, the interferon hybrid portion of the interferon hybrid albumin fusion protein can include additional combinations of α-α interferon hybrids, α-β interferon hybrids, and β-α interferon hybrids. In further embodiments, the interferon hybrid portion of the interferon hybrid albumin fusion protein can be modified to include mutations, substitutions, deletions, or additions to the amino acid sequence of the interferon hybrid. Such modifications to the interferon hybrid albumin fusion protein can be made, for example, to improve the level of production, increase stability, increase or decrease activity, or confer new biological properties.

  The above-described interferon hybrid albumin fusion proteins are encompassed by the present invention, as are host cells and vectors containing polynucleotides encoding such polypeptides. In one embodiment, the interferon hybrid albumin fusion protein encoded by the polynucleotide described above has an extended shelf life. In further embodiments, the interferon hybrid albumin fusion protein encoded by the polynucleotide described above has a longer serum half-life and / or in solution (or pharmaceutical composition) than the corresponding unfused interferon hybrid molecule in vitro and / or in vivo. More stable activity at medium).

  In another non-limiting example, a “therapeutic protein” is a protein having a biological activity, particularly a biological activity useful for treating, preventing or ameliorating a disease. A non-comprehensive list of biological activities that can be retained by therapeutic proteins includes enhancing immune response, promoting angiogenesis, inhibiting angiogenesis, regulating endocrine function, regulating hematopoietic function, neural intestine Biology described in the following biological proteins, described in the “Biological Activity” section below and / or in Table 1 (column 2): Including any one or more of specific activities.

  As used herein, “therapeutic activity” or “activity” means that the effect is consistent with a desired therapeutic outcome in humans or desired in a non-human mammal or other species or organism. An activity that matches the effect can be obtained. The therapeutic activity can be measured in vivo or in vitro. For example, the desired effect can be assayed in cell culture. As an example, when EPO is a therapeutic protein, as described in Example 8, the effect of EPO on cell proliferation can be used as an endpoint where therapeutic activity is measured. Such in vitro or assay or cell culture assays are commercially available for many therapeutic proteins, as described in the art. Examples of assays include, but are not limited to, those described herein in the Examples section or in the “Exemplary Activity Assay” column (column 3) of Table 1.

  Therapeutic proteins (eg, cell surface proteins and secreted proteins) corresponding to the therapeutic protein portion of the albumin fusion proteins of the invention are often modified by the attachment of one or more oligosaccharide groups. Modifications referred to as glycosylation can dramatically affect the physical properties of proteins and can be important in protein stability, secretion and localization. Glycosylation occurs at specific locations along the polypeptide backbone. There are usually two major types of glycosylation: glycosylation characterized by O-linked oligosaccharides (which are linked to serine or threonine residues); and N-linked oligosaccharides. Glycosylated (which is linked to an asparagine residue in the Asn-X-Ser or Asn-X-Thr sequence, where X can be any amino acid except proline). N-acetylneuraminic acid (also known as sialic acid) is usually the terminal residue of both N-linked and O-linked oligosaccharides. Variables (eg, protein structure and cell type) affect the number and nature of carbohydrate units in the chain at different glycosylation sites. Glycosylated isomers are also common at the same site within a given cell type.

  For example, some forms of human interferon are glycosylated. Natural human interferon-α2 is O-glycosylated at threonine 106, and interferon-α14 is N-glycosylated at asparagine 72. (Adolf et al., J. Biochem 276: 511 (1991); Nyman TA et al., J. Biochem 329: 295 (1998)). The oligosaccharides in asparagine 80 of natural interferon-β1α can play important factors in protein solubility and stability, but are not essential for its biological activity. This allows the generation of non-glycosylated analogs (interferon-βlb) that have been engineered by sequence modifications to enhance stability (Hosoi et al., J. Interferon Res. 8: 375 (1988; Karpusas et al., Cell Mol Life Sci 54: 1203 (1998); Knight, J. Interferon Res. 2: 421 (1982); Runkel et al., Pharm Res 15: 641 (1998); Lin, Dev. Biol. Stand. 96: 97 (1998). )) Interferon-γ contains two N-linked oligosaccharide chains at positions 25 and 97, both of which are important for the efficient formation of bioactive recombinant proteins, and the pharmacokinetic properties of the proteins Against (Sareneva et al., Eur. J. Biochem 242: 191 (1996); Sareneva et al., Biochem J. 303: 831 (1994); Sareneva et al., J. Interferon Res. 13: 267 (1993)). O-linked glycosylation and N-linked glycosylation also exist, for example, in human erythropoietin, while N-linked glycosylation exists at asparagine residues located at positions 24, 38, and 83, while O-linked glycosylation is present at the serine residue located at position 126 (Lai et al., J. Biol. Chem. 261 3116 (1986); Brody et al., Arch. Biochem. Biophys. 265: 329 (1988). ) .

  Glycosylation of the EPO albumin fusion protein can affect the activity and / or stability of the EPO albumin fusion protein. The EPO portion of the albumin fusion protein can contain three N-binding sites for glycosylation, each of which can have one tetraantennary structure. If an EPO albumin fusion protein is glycosylated, the half-life of the molecule can be increased. In one embodiment, the EPO albumin fusion protein is glycosylated. In another embodiment, the EPO albumin fusion protein is hyperglycosylated.

  One type of sugar commonly found in oligosaccharides is sialic acid. Each tetraantennary structure of the N-linked glycosylation site of EPO can have four sialic acid residues. Thus, in a preferred embodiment, the EPO albumin fusion protein is glycosylated with a carbohydrate group comprising sialic acid. In a further embodiment, the EPO albumin fusion protein comprises a fully sialylated EPO protein comprising a sialic acid residue / tetraantennary structure / site with a sialic acid to protein molar ratio of 12: 1 or greater. Including. In an alternative embodiment, the EPO albumin fusion protein comprises a hypersialylated EPO protein. Here, one, two, or three sialic acid residues are attached to each tetraantennary structure / site at a molar ratio of sialic acid to protein of less than 12: 1.

  Two types of sialic acid that can be used in sialylation of EPO albumin fusion proteins are N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc). In a preferred embodiment, the hypersialylated EPO albumin fusion protein comprises Neu5Ac. More preferably, the total sialic acid content of the excess sialylated EPO albumin fusion protein is at least 97% Neu5Ac. Most preferred is an EPO albumin fusion protein structure with little or no Neu5Gc.

  Preferably, the albumin EPO fusion protein has at least 4 molar sialylation, more preferably at least 8-9 molar sialylation. Further embodiments include 4 moles sialylation, 5 moles sialylation, 6 moles sialylation, 7 moles sialylation, 8-9 moles sialylation, 8 moles sialylation, 9 moles sialylation, 10 moles, Including albumin EPO fusion proteins with molar sialylation, 11 molar sialylation, or 12 molar sialylation.

  The degree of protein sialylation changes the charge of the protein and its retention time on the chromatography column. Thus, the specific column chromatography steps used in the purification process can be used to monitor or enrich for excess sialylated EPO albumin fusion protein. In preferred embodiments, the amount of sialylation can be monitored by HPLC chromatography. In further embodiments, steps in the purification process of an EPO albumin fusion can be used to enrich for excess sialylated EPO albumin fusion protein. In a preferred embodiment, the purification steps that can be used to enrich for excess sialylated EPO albumin fusion protein are for the butyl sepharose FF purification step to remove virus particles due to high ammonium salts and the final purification step. Includes hydroxyapatite chromatography at pH 6.8.

  Therapeutic protein portions of the albumin fusion proteins of the present invention, and the therapeutic proteins corresponding to analogs and variants thereof, are glycosylated at one or more sites by the host cell in which they are expressed or their expression. Due to other conditions, they can be modified to change as a result of manipulation of their nucleic acid sequences. For example, glycosylated isomers can be generated by removing or introducing glycosylation sites (eg, by substitution or deletion of amino acid residues (eg, substitution of glutamine for asparagine)) or non-glycosylated groups The replacement protein can be produced by expressing the protein in a host cell that does not glycosylate the protein (eg, in E. coli or glycosylation deficient yeast). These approaches are described in more detail below and are known in the art.

  Therapeutic proteins, in particular the therapeutic proteins listed in Table 1, and their nucleic acid and amino acid sequences are well known in the art and are published in public databases (eg, Chemical Abstracts Services Databases (eg, CAS Registry), GenBank, ) And subscription databases (eg, GenSeq (eg, Derwent). Exemplary nucleotide sequences of therapeutic proteins that can be used to derive the polynucleotides of the invention are listed in Table 2. Shown in column 7 “SEQ ID NO: X.” The sequence shown as SEQ ID NO: X can be a wild-type polynucleotide sequence encoding a given therapeutic protein (eg, either full-length or mature), or how many The sequence may be a variant of the wild-type polynucleotide sequence (eg, a polynucleotide encoding a wild-type therapeutic protein), wherein the DNA sequence of the polynucleotide is, for example, a particular species Or a polynucleotide encoding a variant of a wild type therapeutic protein variant (ie, a site-directed variant; an allelic variant). It is well within the ability of one skilled in the art to use the sequence shown as SEQ ID NO: X to obtain the construct described in the same row. For example, if SEQ ID NO: X corresponds to a full-length protein, but only part of that protein is used to generate a specific CID, a specific fragment is amplified and cloned into an appropriate vector Thus, it is within the skill of the artisan to rely on molecular biological techniques (eg, PCR).

  Additional therapeutic proteins corresponding to the albumin fusion proteins of the present invention in the therapeutic protein portion are the therapeutic proteins or peptides disclosed in the column of “Therapeutic protein X” in Table 1 (first column), or fragments or Non-limiting examples include one or more of variable variants.

  Table 1 provides a non-exhaustive list of therapeutic proteins corresponding to the therapeutic protein portion of the albumin fusion proteins of the invention, or albumin fusion proteins encoded by the polynucleotides of the invention. The first column “Therapeutic Protein X” discloses therapeutic protein molecules, followed by the scientific and trade names of the proteins comprising or consisting of the therapeutic protein molecules or fragments or variants thereof in parentheses. including. “Therapeutic protein X” as used herein refers to either an individual therapeutic protein molecule or the entire group of therapeutic proteins associated with a given therapeutic protein molecule disclosed in this column. Say. The “Biological Activity” column (second column) describes the biological activity associated with the therapeutic protein molecule. The third column, “Exemplary Activity Assay”, is used to test the therapeutic and / or biological activity of an albumin fusion protein comprising a therapeutic protein X, or therapeutic protein X (or fragment thereof) portion. References describing assays that can be performed are provided. Each of the references cited in the “Exemplary Activity Assay” column is specifically described in the reference to assay the corresponding biological activity shown in the “Biological Activity” column of Table 1. The description of each activity assay is incorporated herein by reference in its entirety (see, eg, those materials and methods). The fifth column “Preferred indication Y” is a disease, disorder, and that can be treated, prevented, diagnosed, and / or ameliorated by an albumin fusion protein comprising a therapeutic protein X or therapeutic protein X (or fragment thereof) moiety. Describe the status. The “Construct ID” column (column 5) encodes an albumin fusion protein disclosed in Table 2 that encodes an albumin fusion protein comprising or consisting of the referenced therapeutic protein X (or fragment thereof) portion. Provides a link to

  Table 2 provides a non-exhaustive list of polynucleotides of the present invention comprising or consisting of nucleic acid molecules encoding albumin fusion proteins. The fifth column “number of fusions” indicates the number of fusions of each polynucleotide. In the second column, “Construct ID” provides a unique numerical identifier for each polynucleotide of the invention. This construct ID is used to identify a polynucleotide encoding an albumin fusion protein that comprises or consists of a therapeutic protein portion corresponding to a given therapeutic protein X listed in the corresponding row of Table 1. Can be done. Here, the construct ID is listed in the fifth column. The “Construct Name” column (third column) provides the name of a given albumin fusion construct or polynucleotide.

The fourth column “Description” of Table 2 provides a general description of a given albumin fusion construct, and the fifth column “Expression Vector” contains or contains nucleic acid molecules encoding a given albumin fusion protein. The vector in which the polynucleotide consisting of was cloned is listed.
Vectors are known in the art and are either commercially available or described elsewhere. For example, as described in the Examples, (1) a polynucleotide encoding a given albumin fusion protein, (2) a leader sequence, (3) a promoter region, and (4) one or more of the transcription terminators Alternatively, an “expression cassette” consisting of one or more of these can be assembled into a convenient cloning vector followed by replacement vectors such as, for example, expression vectors (eg, yeast expression vectors or mammalian expression vectors). Assembly). In one embodiment, S.I. For expression in C. cerevisiae, an expression cassette containing or consisting of a nucleic acid molecule encoding an albumin fusion protein is cloned into pSAC35. In another embodiment, an expression cassette containing or consisting of a nucleic acid molecule encoding an albumin fusion protein is cloned into pC4 for expression in CHO cells. In further embodiments, a polynucleotide comprising or consisting of a nucleic acid molecule encoding a therapeutic protein portion of an albumin fusion protein is cloned into pC4: HSA. In still further embodiments, an expression cassette comprising or consisting of a nucleic acid molecule encoding an albumin fusion protein is cloned into pEE12 for expression in NS0 cells. Other useful cloning and / or expression vectors are known to those skilled in the art and are within the scope of the present invention.

  The sixth column “SEQ ID NO: Y” provides the full-length amino acid sequence of the albumin fusion protein of the present invention. In most instances, SEQ ID NO: Y represents the unprocessed form of the encoded albumin fusion protein (in other words, SEQ ID NO: Y contains all of the signal sequence, HSA portion, and therapeutic portion encoded by a particular construct. Specifically contemplated by the present invention are all polynucleotides that encode SEQ ID NO: Y. When these polynucleotides are used to express the encoded protein from a cell, the native of the cell The secretory and processing steps of produce a protein that lacks the signal sequences listed in columns 4 and / or 11 of Table 2. The specific amino acid sequences of the listed signal sequences are later described herein. Or is well known in the art, so the most preferred embodiment of the present invention is by cell. The resulting albumin fusion protein (which lacks the leader sequence shown in columns 4 and / or 11 of Table 2), most preferably listed in columns 4 and / or 11 of Table 2. A polypeptide comprising SEQ ID NO: Y without any particular leader sequence, also preferred is a composition comprising these two preferred embodiments, including a pharmaceutical composition, in addition to the fourth column of Table 2 and Relaxing the signal sequence listed in column 11 with a different signal sequence (eg, a signal sequence described later herein to facilitate secretion of the processed albumin fusion protein). Is well within the ability of one skilled in the art.

  The seventh column “SEQ ID NO: X” provides the parental nucleic acid sequence from which a polynucleotide encoding the therapeutic protein portion of a given albumin fusion protein can be obtained. In one embodiment, the parent nucleic acid sequence from which the polynucleotide encoding the therapeutic protein portion of the albumin fusion protein can be obtained comprises the wild type gene sequence encoding the therapeutic protein shown in Table I. In an alternative embodiment, the parent nucleic acid sequence from which the polynucleotide encoding the therapeutic protein portion of the albumin fusion protein can be obtained is a variant or derivative of a wild type gene sequence encoding the therapeutic protein shown in Table I ( For example, a modified wild-type gene sequence encoding a therapeutic protein, optimized with synthetic codons).

  Column 8 “SEQ ID NO: Z” provides a putative translation of the parent nucleic acid sequence (SEQ ID NO: X). This parent sequence is a full-length parent protein used to obtain a particular construct, a mature portion of the parent protein, a variant or fragment of a wild type protein, or an artificial sequence that can be used to create the described construct. possible. One skilled in the art can use this amino acid sequence shown in SEQ ID NO: Z to determine which amino acid residues of an albumin fusion protein encoded by a given construct are provided by a therapeutic protein. Furthermore, it is well within the ability of one skilled in the art to use the sequence shown as SEQ ID NO: Z to obtain the construct described in the same row. For example, if SEQ ID NO: Z corresponds to a full-length protein, but only a portion of that protein is used to generate a specific CID, a specific fragment is amplified and cloned into an appropriate vector Thus, it is within the skill of the artisan to rely on molecular biological techniques (eg, PCR).

  Amplification primers "SEQ ID NO: A" and "SEQ ID NO: B" provided in columns 9 and 10, respectively, are polynucleotides that comprise or consist of nucleic acid molecules that encode the therapeutic protein portion of a given albumin fusion protein Is an exemplary primer used to generate In one embodiment of the present invention, an oligonucleotide primer having the sequence shown in columns 9 and 10 (SEQ ID NO: A and / or B) is the nucleotide sequence provided in column 7 of the corresponding row (sequence A nucleic acid molecule comprising or consisting of number X) is used as template DNA to PCR amplify a polynucleotide encoding the therapeutic protein portion of an albumin fusion protein. PCR methods are well established in the art. Additional useful primer sequences can be readily envisioned and utilized by those skilled in the art.

  In an alternative embodiment, oligonucleotide primers can be used in duplicate PCR reactions to generate mutations in the template DNA sequence. PCR methods are known in the art.

  As shown in Table 3, certain albumin fusion constructs disclosed in this application have been deposited with the ATCC®.

当該分野で公知の技術および本明細書中他の箇所に記載される技術（例えば、実施例４０を参照のこと）により、寄託物から構築された所定のアルブミン融合構築物を回収することが可能である。ＡＴＣＣは、１０８０１ Ｕｎｉｖｅｒｓｉｔｙ Ｂｏｕｌｅｖａｒｄ，Ｍａｎａｓｓａｓ，Ｖｉｒｇｉｎｉａ ２０１１０−２２０９，ＵＳＡに存在する。
ＡＴＣＣへの寄託は、特許手続上の微生物の寄託の国際的承認に関するブタベスト条約の規定に準じてなされた。

It is possible to recover a given albumin fusion construct constructed from the deposit by techniques known in the art and described elsewhere herein (eg, see Example 40). is there. ATCC exists in 10801 University Boulevard, Manassas, Virginia 20110-2209, USA.
Deposits with the ATCC were made in accordance with the provisions of the Butbestos Convention on the international recognition of deposits of microorganisms in patent proceedings.

  In a further embodiment of the invention, or comprising one or more of (1) a polynucleotide encoding a given albumin fusion protein, (2) a leader sequence, (3) a promoter region, and (4) a transcription terminator An “expression cassette” consisting of them can be transferred or “subcloned” from one vector to another. Subcloned fragments can be generated by methods well known in the art, such as PCR amplification (eg, using an oligonucleotide primer having the sequence shown in SEQ ID NO: A or B) and / or restriction enzyme digestion.

  In a preferred embodiment, the albumin fusion protein of the invention corresponds to a therapeutic activity and / or biological activity corresponding to a therapeutic activity and / or a therapeutic protein portion of an albumin fusion protein listed in the corresponding row of Table 1. It may have biological activity of a therapeutic protein. In a further preferred embodiment, the therapeutically active protein part of the albumin fusion protein of the invention is a fragment or variant of the protein encoded by the sequence shown in the column of SEQ ID NO: X in Table 2 and the therapeutic activity of the corresponding therapeutic protein And / or have biological activity.

(Non-human albumin fusion protein of growth hormone)
In one embodiment, in an albumin fusion protein of the invention, one or more serum albumin proteins of a non-human animal species are transferred to one or more growth hormone proteins of the same non-human animal species at their N-terminal or C-terminal. One is fused in tandem and in frame. Non-human serum albumin and growth hormone proteins are well known in the art and are available in public databases. For example, Table 4 shows the accession numbers corresponding to the non-human serum albumin sequences (second column) and non-human growth hormone sequences (third column) found in GenBank. In a preferred embodiment, serum albumin proteins from non-human animal species listed in Table 4 are fused to growth hormone proteins from the same non-human animal species.

  In certain embodiments, in an albumin fusion protein of the invention, one or more Bos taurus serum albumin proteins listed in Table 4, second column are replaced with one or more Bos listed in Table 4, third column. It is fused to taurus growth hormone in tandem and in-frame at its N-terminus or C-terminus.

  Fusion proteins comprising fragments or variants of non-human serum albumin (eg, mature forms of serum albumin) are also encompassed by the present invention. Fusion proteins comprising fragments or variants of non-human growth hormone proteins (eg, mature forms of growth hormone) are also encompassed by the present invention. Preferably, non-human growth hormone fragments and variants retain growth activity.

The polynucleotide of the present invention comprises or consists of one or more nucleic acid molecules encoding the above non-human albumin fusion proteins. For example, the polynucleotide comprises one or more nucleic acid molecules encoding a corresponding non-human animal species non-human growth hormone protein (eg, a growth hormone reference sequence listed in Table 4, Column 3), or Serum albumin proteins from the non-human animal species listed in Table 4, column 1, fused in tandem and in-frame, either 5 'or 3' to the polynucleotides comprising them (
For example, it may comprise or consist of one or more nucleic acid molecules encoding the non-human serum albumin reference sequence listed in Table 4, column 2).

  The above non-human albumin fusion proteins are encompassed by the present invention, as are host cells and vectors containing these polynucleotides. In one embodiment, the non-human albumin fusion protein encoded by the polynucleotide described above has an extended shelf life. In further embodiments, the non-human albumin fusion protein encoded by the polynucleotide described above is longer serum in solution (or pharmaceutical composition) in vitro and / or in vivo than the corresponding non-fusion growth hormone molecule. Has a half-life and / or more stable activity.

  The invention also encompasses methods for preventing, treating, or alleviating a disease or disorder in a non-human animal species. In certain embodiments, the invention encompasses a method of treating a veterinary disease or disorder, wherein the method includes treating the disease or disease in a non-human animal species for which such treatment, prevention, or alleviation is desired. Administering an albumin fusion protein of the invention comprising a growth hormone moiety corresponding to a growth hormone protein (or fragment or variant thereof) in an amount effective to treat, prevent, or alleviate the disorder. Veterinary diseases and / or disorders that can be treated, prevented, or alleviated include growth disorders (eg, pituitary dwarfism), skin pain, obesity, growth hormone responsive skin disease, dilated cardiomyopathy, diet Disorders, reproductive disorders, and endocrine disorders.

  The non-human albumin fusion proteins of the present invention can also be used to facilitate the treatment of skin wounds, corneal damage, fractures, and joint, tendon, or ligament damage.

  The non-human albumin fusion proteins of the present invention can also be used to increase milk production in lactating animals. In a preferred embodiment, the milking animal is a dairy cow.

  The non-human albumin fusion proteins of the present invention can also be used to improve the physical condition in aging animals.

  The non-human albumin fusion proteins of the present invention can also be used to increase conception, pregnancy rate, and reproductive success in domestic animals.

  The non-human albumin fusion protein of the present invention is also used to improve the lean to fat ratio, improve appetite, and increase body size and growth rate in animals that are elevated in wasting. obtain.

（ポリペプチドおよびポリヌクレオチドのフラグメントおよび改変体）
（フラグメント）
本発明はさらに、表１に記載される治療タンパク質、アルブミンタンパク質、および／または本発明のアルブミン融合タンパク質のフラグメントに関する。

(Polypeptide and polynucleotide fragments and variants)
(Fragment)
The present invention further relates to therapeutic proteins, albumin proteins, and / or fragments of albumin fusion proteins of the present invention described in Table 1.

  The invention also relates to polynucleotides that encode the therapeutic proteins listed in Table 1, albumin proteins, and / or fragments of albumin fusion proteins of the invention.

  Even if deletion of one or more amino acids from the N-terminus of the protein results in alteration or loss of one or more biological functions of the therapeutic protein, albumin protein and / or albumin fusion protein of the invention, etc. Can still retain therapeutic and / or functional activity (eg, biological activity, ability to multimerize, ability to bind ligand). For example, the ability of a polypeptide with an N-terminal deletion to induce and / or bind an antibody that recognizes the complete or mature form of this polypeptide removes less than most of the residues of the complete polypeptide from the N-terminus. In general, it is retained. Whether a particular polypeptide lacking the N-terminal residue of a complete polypeptide retains such immunological activity is determined by routine methods described herein and otherwise in the art. It can be easily determined by known methods. It is unlikely that a mutein lacking many N-terminal amino acid residues can retain some biological or immunological activity. Indeed, peptides composed of as many as 6 amino acid residues can often elicit an immunological response.

  Accordingly, therapeutic protein fragments corresponding to the therapeutic protein portion of an albumin fusion protein of the invention include full-length proteins and one or more residues of reference polypeptides (ie, therapeutic proteins shown in Table 1 or Table 2). And a polypeptide deleted from the amino terminus of the amino acid sequence of the albumin fusion protein encoded by the polynucleotide or albumin fusion construct). In particular, the N-terminal deletion may be described by the general formulas m to q, where q is a reference polypeptide (eg, a therapeutic protein shown in Table 1 or a therapeutic protein portion or table of an albumin fusion protein of the invention). 2 is a whole integer indicating the total number of amino acid residues in the therapeutic protein portion of the albumin fusion protein encoded by the polynucleotide or albumin fusion construct described in 2, and m is any integer in the range of 2 to q-6 Is defined as Polynucleotides encoding these polypeptides are also encompassed by the present invention.

  Further, fragments of the serum albumin polypeptide corresponding to the albumin protein portion of the albumin fusion protein of the invention include the full-length protein and one or more residues described in the reference polypeptide (ie, serum albumin or Table 2). A polypeptide deleted from the amino terminus of the amino acid sequence of the albumin fusion protein encoded by the polynucleotide or albumin fusion construct). In a preferred embodiment, the N-terminal deletion can be described by the general formula m-585, where 585 is a whole integer indicating the total number of amino acid residues in mature human serum albumin (SEQ ID NO: 1038), and m Is defined as any integer in the range of 2 to 579. Polynucleotides encoding these polypeptides are also encompassed by the present invention. In a further embodiment, the N-terminal deletion can be described by the general formula m-609, where 609 is a whole integer indicating the total number of amino acid residues in full-length human serum albumin (SEQ ID NO: 1094), and m Is defined as any integer in the range of 2 to 603. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

  Further, fragments of the albumin fusion protein of the present invention include full-length albumin fusion proteins and albumin fusions in which one or more residues are encoded by an albumin fusion protein (eg, a polynucleotide or albumin fusion construct described in Table 2). Or a polypeptide deleted from the amino terminus of an albumin fusion protein having the amino acid sequence disclosed in column 6 of Table 2). In particular, the N-terminal deletion can be described by the general formulas m to q, where q is a whole integer indicating the total number of amino acid residues in the albumin fusion protein and m is in the range of 2 to q-6. Defined as an arbitrary integer. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

  As noted above, deletion of one or more amino acids from the N-terminus or C-terminus of a reference polypeptide (eg, therapeutic protein; serum albumin protein; or albumin fusion protein of the invention) results in one or more of the proteins Other functional activities (eg, biological activity, ability to multimerize, ability to bind ligand) and / or therapeutic activity may still be retained, even if alterations or loss of the biological function of. For example, the ability of a polypeptide with a C-terminal deletion to induce and / or bind an antibody that recognizes the complete or mature form of the polypeptide is less than most of the residues of the complete or mature polypeptide. It is generally retained when removed from the C-terminus. Whether a particular polypeptide lacking the N-terminal residue and / or C-terminal residue of a reference polypeptide retains therapeutic activity is actually determined by the conventional methods described herein and / or Or else it can be easily determined by methods known in the art.

  The invention further provides a therapeutic protein portion of an albumin fusion protein of the invention (eg, a therapeutic protein shown in Table 1, or a therapeutic protein portion of an albumin fusion protein encoded by a polynucleotide or albumin fusion construct shown in Table 2). ) In which one or more residues are deleted from the carboxy terminus of the amino acid sequence of the therapeutic protein. In particular, the C-terminal deletion may be described by general formulas 1-n, where n is any whole integer ranging from 6 to q-1 and q is a reference polypeptide (eg, in Table 1). All integers representing the total number of amino acid residues in the indicated therapeutic protein or therapeutic protein portion of the albumin fusion protein encoded by the polynucleotide or albumin fusion construct described in Table 2. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

  Furthermore, the present invention corresponds to an albumin protein portion of an albumin fusion protein of the present invention (eg, albumin protein portion of an albumin fusion protein encoded by serum albumin or a polynucleotide or albumin fusion construct described in Table 2). Polypeptides in which one or more residues are deleted from the carboxy terminus of the amino acid sequence of albumin protein are provided. In particular, the C-terminal deletion may be described by general formulas 1-n, where n is any whole integer ranging from 6-584, where 584 is mature human serum albumin (SEQ ID NO: 1038) It is a whole integer representing the total number of amino acid residues in the -1. Polynucleotides encoding these polypeptides are also encompassed by the present invention. In particular, the C-terminal deletion may be described by general formulas 1-n, where n is any whole integer ranging from 6-608, where 608 is an amino acid in serum albumin (SEQ ID NO: 1094) All integers representing the total number of residues minus 1. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

  Furthermore, the present invention provides a polypeptide in which one or more residues are deleted from the carboxy terminus of the albumin fusion protein of the present invention. In particular, the C-terminal deletion may be described by general formulas 1 to n, where n is any whole integer ranging from 6 to q-1 and q is an amino acid in the albumin fusion protein of the invention A whole integer representing the total number of residues. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

Further, any of the N-terminal deletions or C-terminal deletions described above can be combined to produce N-terminal and C-terminal deletion reference polypeptides. The invention also provides polypeptides in which one or more amino acids have been deleted from both the amino terminus and the carboxyl terminus. This polypeptide may be a reference polypeptide (eg, a therapeutic protein shown in Table 1 or a therapeutic protein portion of an albumin fusion protein of the invention, or a therapeutic protein encoded by a polynucleotide or albumin fusion construct described in Table 2. A portion, or serum albumin (eg, SEQ ID NO: 1038), or an albumin protein portion of an albumin fusion protein of the invention, or an albumin protein portion encoded by a polynucleotide or albumin fusion construct described in Table 2, or an albumin fusion protein Or residues m to n of an albumin fusion protein encoded by a polynucleotide or albumin fusion construct of the invention).
Here, n and m are integers as described above. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

  The application of the invention also applies to the reference polypeptide sequences shown herein (the therapeutic protein shown in Table 1, or the therapeutic protein portion of the albumin fusion protein of the invention, or the polynucleotide or albumin described in Table 2). The therapeutic protein portion encoded by the fusion construct, or serum albumin (eg, SEQ ID NO: 1038), or the albumin protein portion of the albumin fusion protein of the invention, or the polynucleotide or albumin fusion construct described in Table 2 Albumin protein portion, or albumin fusion protein, or albumin fusion protein encoded by a polynucleotide or albumin fusion construct of the invention) and at least 80%, 85%, 90%, 95%, 96% 97%, protein 98%, or 99% identical to the polypeptide or to a fragment thereof. In a preferred embodiment, the application is at least 80%, 85%, 90%, 95%, 96%, 97% with a reference polypeptide having an N-terminal deletion and C-terminal deletion amino acid sequence as described above. It relates to a protein comprising 98% or 99% identical polypeptides. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

  Preferred polypeptide fragments of the invention comprise an amino acid sequence that exhibits therapeutic and / or functional activity (eg, biological activity) of the polypeptide sequence of the therapeutic protein or serum albumin protein whose amino acid sequence is the fragment, or Consist of them.

  Other preferred polypeptide fragments are biologically active fragments. Biologically active fragments are those that exhibit activity that is similar, but not necessarily identical, to the activity of the polypeptides of the invention. The biological activity of this fragment may include an improved desired activity or a reduced undesirable activity.

(Modified)
“Variant” refers to a polynucleotide or nucleic acid that differs from a reference nucleic acid or polypeptide but retains their essential properties. In general, variants are generally very similar and in many regions are identical to a reference nucleic acid or polypeptide.

  As used herein, “variant” refers to a therapeutic protein (see, eg, the “Therapeutic” column of Table 1), albumin protein, and / or albumin fusion protein, respectively. Albumin of the present invention that is different but retains at least one functional and / or therapeutic property thereof as described elsewhere herein or otherwise known in the art It refers to the therapeutic protein portion of the fusion protein, the albumin portion of the albumin fusion protein of the invention, or the albumin fusion protein of the invention. In general, the variants are very similar overall and in many regions the therapeutic protein corresponding to the therapeutic protein portion of the albumin fusion protein, the albumin corresponding to the albumin protein portion of the albumin fusion protein It is identical to the amino acid sequence of the protein and / or albumin fusion protein. Nucleic acids encoding these variants are also encompassed by the present invention.

  The present invention also includes, for example, the amino acid sequence of a therapeutic protein corresponding to the therapeutic protein portion of an albumin fusion protein of the present invention (eg, the therapeutic protein disclosed in Table 1: the amino acid sequence of X; or Tables 1 and 2 Of the therapeutic protein portion of an albumin fusion protein encoded by the polynucleotide or albumin fusion construct described in or a fragment or variant thereof), of the albumin protein corresponding to the albumin protein portion of the albumin fusion protein of the invention An amino acid sequence (eg, the amino acid sequence of the albumin protein portion of an albumin fusion protein encoded by a polynucleotide or albumin fusion construct described in Tables 1 and 2; Acid sequence; or fragments or variants thereof) and / or the amino acid sequence of the albumin fusion protein, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least It relates to a protein comprising or consisting of 98%, at least 99% or at least 100% identical amino acid sequences. Fragments of these polypeptides are also provided (eg, the fragments described herein). Additional polypeptides included in the present invention hybridize to filter-bound DNA under stringent hybridization conditions (eg, at about 45 ° C. in 6 × sodium chloride / sodium citrate (SSC)) Then wash one or more times in 0.2 × SSC, 0.1% SDS at about 50-65 ° C.) under highly stringent conditions (eg 6 × sodium chloride / sodium citrate (SSC)) Hybridization at about 45 ° C. to filter-bound DNA, followed by one or more washes in 0.1 × SSC, 0.2% SDS at about 68 ° C.), or as known to those skilled in the art Under certain other stringent hybridization conditions (eg, Ausubel, FM et al., 1989, Curren see Protocols in Molecular Biology, Green publishing associates, Inc. and John Wiley & Sons Inc. New York, pages 6.3.1-6.3.6 and 2.10.3). A polypeptide encoded by a polynucleotide that hybridizes to the complement of a nucleic acid molecule that encodes. Polynucleotides encoding these polypeptides are also included in the present invention.

  The subject polypeptide sequence may include up to 5 amino acid changes for each 100 amino acids of the query amino acid sequence, eg, by a polypeptide having an amino acid sequence that is at least 95% “identical” to the query amino acid sequence Except that the amino acid sequence of the subject polypeptide is identical to the query sequence. In other words, to obtain a polypeptide having an amino acid sequence that is at least 95% identical to the query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted or deleted It may be substituted with an amino acid. These modifications of the reference sequence can occur at the amino-terminal position or the carboxy-terminal position of the reference amino acid sequence, or individually between residues in the reference sequence or in one or more consecutive groups within the reference sequence It can occur anywhere between their end positions interspersed with either.

  In practice, any particular polypeptide may be at least relative to the amino acid sequence of, for example, an albumin fusion protein of the invention or a fragment thereof (eg, a therapeutic protein portion of an albumin fusion protein or an albumin portion of an albumin fusion protein). Whether 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% are identical can conventionally be determined using known computer programs. A preferred method (also called global sequence alignment) for determining the best overall match between a query sequence (sequence of the invention) and a subject sequence is Brutlag et al. (Comp. App. Biosci. 6: 237-245 (1990)) and can be determined using a FASTDB computer program. In sequence alignment, the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The above overall sequence alignment results are given in percent identity. Preferred parameters used for FASTDB amino acid alignment are: Matrix = PAM 0, k-tuple = 2, Mismatch Penalty = 1, Joining Penalty = 20, Randomization Group Length = 0, Cutoff Score = 1, Window Size length, Window Size = Gap Penalty = 5, Gap Size Penalty = 0.05, Window Size = 500, or the length of the target amino acid sequence (whichever is shorter).

  If the subject sequence is shorter than the query sequence (not due to an internal deletion) due to an N-terminal or C-terminal deletion, a manual correction must be made to the result. This is because the FASTDB program does not consider N-terminal cleavage and C-terminal truncation of the subject sequence when calculating the overall percent identity. For subject sequences that are truncated at the N-terminus and C-terminus relative to the query sequence, the percent identity is the percent of total bases in the query sequence that does not match / align with the corresponding subject residue. And is corrected by calculating the number of residues in the query sequence that are C-terminal. Whether a residue is matched / aligned is determined by the results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity calculated using the specified parameters by the FASTDB program described above to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present invention. Only residues at the N-terminal and C-terminal side of the subject sequence that do not match / align with the query sequence are considered for the purpose of manually adjusting the percent identity score. That is, only the query residues are located outside the farthest N-terminal and C-terminal residues of the subject sequence.

  For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. Deletions occur at the N-terminus of the subject sequence and therefore the FASTDB alignment does not show a match / alignment of the first 10 residues at the N-terminus. The 10 unpaired residues correspond to 10% of the sequence (number of residues at non-matching N- and C-termini / total number of residues in the query sequence), so it is calculated by the FASTDB program. 10% is subtracted from the percent identity score. If the remaining 90 residues are perfectly matched, the final percent identity is 90%. In another example, a 90 residue subject sequence is compared to a 100 residue query sequence. In this case, the deletion is an internal deletion, so there are no N-terminal or C-terminal residues of the subject sequence that do not match / align with the query sequence. In this case, the percent identity calculated by FASTDB is not manually corrected. Again, only the residue positions outside the N- and C-termini of the subject sequence that do not match / align with the query sequence shown in the FASTDB alignment are manually corrected. No other manual correction is made for the purposes of the present invention.

  A variant has at least 75% (preferably at least about 80%, at least 90%, at least 95%, or at least 99%) sequence identity with the length of a normal HA or therapeutic protein of the same length as the variant. Usually have. Nucleotide or amino acid sequence level homology or identity is determined by BLAST (Basic Local Alignment Search) analysis using the algorithm used by the programs blastp, blastn, blastx, tblastn and tblastx tailored for sequence similarity searches. (Karlin et al., Proc. Natl. Acad. Sci. USA 87: 2264-2268 (1990) and Altschul, J. Mol. Evol. 36: 290-300 (1993) (incorporated by reference)). .

  The approach used by the BLAST program first considers similar segments between the query sequence and the database sequence, then evaluates the statistical significance of all matches identified, and finally significance Only matches that satisfy a preselected threshold of. See Altschul et al. (Nature Genetics 6: 119-129 (1994)) (all incorporated by reference) for a discussion of basic issues in similarity searching of sequence databases. The search parameters for histogram, description, alignment, expected value (ie, statistically significant threshold for reporting matches against the database sequence), cutoff, matrix and filter are the default settings. The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et al., Proc. Natl. Acad. Sci. USA 89: 10915-10919 (1992), all incorporated by reference) It is. For blastn, the scoring matrix is set by the ratio of M (ie, the reward score for a pair of matched residues) to N (ie, the penalty score for mismatched residues), where The default values for M and N are 5 and -4, respectively). The four blastn parameters can be adjusted as follows: Q = 10 (gap creation penalty); R = 10 (gap extension penalty); wink = 1 (word hit at every wink position along the query sequence) And gapw = 16 (sets the window width in which the gap alignment is generated). The equivalent Blastp parameter settings were Q = 9; R = 2; wink = 1; and gapw = 32. Bestfit comparison between sequences available in GCG package version 10.0 uses DNA parameters GAP = 50 (gap creation penalty) and LEN = 3 (gap extension penalty), and the equivalent setting in protein comparison is GAP = 8 and LEN = 2.

  The polynucleotide variants of the invention may include changes in the coding region, non-coding region, or both. Particularly preferred are polynucleotide variants that contain changes that produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred. Further, in any combination, less than 50, less than 40, less than 30, less than 20, less than 10, or 5-50, 5-25, 5-10, 1-5, or 1 Also preferred are polypeptide variants in which two amino acids are substituted, deleted, or added. Polynucleotide variants are for a variety of reasons (eg, to optimize codon expression for a particular host (changing codons in human mRNA to preferred codons for yeast or bacterial hosts such as E. coli)). Can be generated.

  In a preferred embodiment, the polynucleotide of the invention encoding the albumin portion of an albumin fusion protein is optimized for expression in yeast or mammalian cells. In a further preferred embodiment, the polynucleotide of the invention encoding the therapeutic protein portion of an albumin fusion protein is optimized for expression in yeast or mammalian cells. In still further preferred embodiments, the polynucleotide encoding the albumin fusion protein of the invention is optimized for expression in yeast or mammalian cells.

  In an alternative embodiment, the codon optimized polynucleotide encoding the therapeutic protein portion of the albumin fusion protein is a wild-type encoding therapeutic protein under stringent hybridization conditions as described herein. Does not hybridize to type polynucleotides. In further embodiments, the codon-optimized polynucleotide that encodes the albumin portion of the albumin fusion protein does not hybridize to the wild-type polynucleotide that encodes the albumin protein under the stringent hybridization conditions described herein. . In another embodiment, the codon-optimized polynucleotide encoding an albumin fusion protein is a wild-type polynucleotide encoding a therapeutic protein moiety or an albumin protein moiety under stringent hybridization conditions described herein. Does not hybridize to

  In further embodiments, the polynucleotide encoding the therapeutic protein portion of the albumin fusion protein does not include or consist of the naturally occurring sequence of the therapeutic protein. In further embodiments, the polynucleotide encoding the albumin protein portion of the albumin fusion protein does not comprise or consist of the naturally occurring sequence of the albumin protein. In an alternative embodiment, the polynucleotide encoding the albumin fusion protein does not comprise or consist of the therapeutic protein portion or the naturally occurring sequence of the albumin protein portion.

  Naturally occurring variants are referred to as “allelic variants” and refer to one of several alternative forms of a gene occupying a given locus on an organism's chromosome. (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985)). These allelic variants can vary at either the polynucleotide level and / or the polypeptide level and are included in the present invention. Alternatively, non-naturally occurring variants can be produced by mutagenesis techniques or by direct synthesis.

Using known methods of protein engineering and recombinant DNA technology, variants can be made to improve or alter the properties of the polypeptides of the invention. For example, one or more amino acids can be deleted from the N-terminus or C-terminus of a polypeptide of the invention without substantial loss of biological function. As an example, Ron et al. (J. Biol. Chem. 268: 2984-2988 (1993)) demonstrated heparin binding activity even after deletion of 3, 8, or 27 amino-terminal amino acid residues. A variant KGF protein was reported.
Similarly, interferon gamma showed up to 10 times higher activity after deletion of 8-10 amino acid residues from the carboxy terminus of the protein (Dobeli et al., J. Biotechnology 7: 199-216). (1988)).

  Furthermore, extensive evidence demonstrates that variants often retain activity similar to the biological activity of naturally occurring proteins. For example, Gayle and coworkers (J. Biol. Chem. 268: 22105-22111 (1993)) performed extensive mutational analysis of the human cytokine IL-1a. They used random mutagenesis to create over 3,500 individual IL-1a variants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were tested at all possible amino acid positions. The researchers found that “the majority of molecules can be altered with little effect on either [binding or biological activity]”. In fact, of the over 3,500 nucleotide sequences tested, only 23 unique amino acid sequences produced proteins that were significantly different in activity from the wild type.

  Furthermore, even if deletion of one or more amino acids from the N-terminus or C-terminus of the polypeptide results in an improvement or loss of one or more biological functions, other biological activities are still retained. obtain. For example, the ability of deletion variants to induce and / or bind antibodies that recognize the secreted form retains when fewer than a majority of the secreted form residues are removed from the N-terminus or C-terminus. It seems to be done. Whether a particular polypeptide lacking the N-terminal or C-terminal residue of a protein retains such immunogenic activity must be determined by conventional methods described herein, and otherwise. It can be easily determined by conventional methods known in the art.

  Accordingly, the present invention further includes polypeptide variants that have functional activity (eg, biological activity and / or therapeutic activity). In one embodiment, the invention provides for a functional activity corresponding to one or more biological and / or therapeutic activities of a therapeutic protein corresponding to a therapeutic protein portion of an albumin fusion protein (eg, biological Variants of albumin fusion proteins having activity and / or therapeutic activity) are provided. In another embodiment, the invention provides a functional activity corresponding to one or more biological and / or therapeutic activities of a therapeutic protein corresponding to a therapeutic protein portion of an albumin fusion protein (eg, biological activity and Variants of albumin fusion proteins having (or therapeutic activity) are provided. Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art that have little effect on activity. Polynucleotides encoding such variants are also included in the present invention.

  In preferred embodiments, the variants of the invention have conservative substitutions. By “conservative substitution” is intended exchange within the group as follows: substitution of aliphatic or hydrophobic amino acids Ala, Val, Leu and Ile; substitution of hydroxyl residues Ser and Thr; acidic residue Asp and Glu substitution; substitution of amide residues Asn and Gln, substitution of basic residues Lys, Arg, and His; substitution of aromatic residues Phe, Tyr, and Trp, and small size amino acids Ala, Ser, Thr, Met and Gly substitutions.

  Guidance on how to make phenotypically silent amino acid substitutions is provided, for example, in Bowie et al., “Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substations”, Science 247: 1306-1310 (1990). The authors then point out that there are two main strategies for studying the tolerance of amino acid sequences to changes.

  The first strategy takes advantage of the tolerance of amino acid substitution by natural selection during the course of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids appear to be important for protein function. In contrast, amino acid positions whose substitutions have been tolerated by natural selection indicate that these positions are not critical to protein function. Thus, positions that tolerate amino acid substitutions can be altered while still maintaining the biological activity of the protein.

  The second strategy uses genetic engineering to introduce amino acid changes at specific positions in the cloned gene to identify regions important for protein function. For example, site-directed mutagenesis or alanine scanning mutagenesis (introduction of one alanine mutation for each residue in the molecule) can be used. See Cunningham and Wells, Science 244: 1081-1085 (1989). The resulting mutant molecules can then be tested for biological activity.

  As the authors mention, these two strategies revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes appear to be tolerated at specific amino acid positions in the protein. For example, amino acid residues that are almost buried (within the tertiary structure of the protein) require nonpolar side chains, but the characteristics of surface side chains are generally rarely conserved. In addition, tolerated conservative amino acid substitutions include substitution of aliphatic or hydrophobic amino acids Ala, Val, Leu, and Ile; substitution of hydroxyl residues Ser and Thr; substitution of acidic residues Asp and Glu; Substitution of Asn and Gln of amide residues, substitution of basic residues Lys, Arg, and His; substitution of aromatic residues Phe, Tyr, and Trp, and small size amino acids Ala, Ser, Thr, Includes substitutions for Met and Gly. In addition to conservative amino acid substitutions, the variants of the present invention may include (i) substitution of one or more non-conservative amino acid residues (where the amino acid residues to be substituted are amino acids encoded by the genetic code) Or (ii) a polypeptide comprising a substitution of one or more amino acid residues having a substituent, or (iii) another compound (e.g., A polypeptide fused or chemically conjugated with a compound (eg, polyethylene glycol) to increase the stability and / or solubility of the polypeptide, (iv) an additional amino acid (eg, an IgG Fc fusion region peptide) A polypeptide comprising Such variant polypeptides are deemed to be within the scope of those skilled in the art from the teachings herein.

  For example, polypeptide variants that include amino acid substitutions of charged amino acids with other charged amino acids or neutral amino acids may produce proteins with improved properties (eg, less aggregation). Aggregation of pharmaceutical formulations results in both decreased activity and increased clearance due to the aggregate's immunogenic activity. Pinckard et al., Clin. Exp. Immunol. 2: 331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev. See Therapeutic Drug Carrier Systems 10: 307-377 (1993).

In certain embodiments, a polypeptide of the invention comprises or consists of an amino acid sequence of an albumin fusion protein, a fragment or variant of an amino acid sequence of a therapeutic protein and / or human serum albumin, wherein This fragment or variant has additions, substitutions of 1-5, 5-10, 5-25, 5-50, 10-50, or 50-150 amino acid residues when compared to a reference amino acid sequence, And / or have a deletion. In preferred embodiments, the amino acid substitution is conservative.
Nucleic acids encoding these polypeptides are also included in the present invention.

  The polypeptides of the present invention may be composed of amino acids linked to each other by peptide bonds or modified peptide bonds, ie, peptide isosteres, and may include amino acids other than the 20 amino acids encoded by the gene. The polypeptide can be modified either by natural processes such as post-translational processing or by chemical modification techniques well known in the art. Such modifications are well documented in basic texts and in more detailed research papers as well as many research literature. Modifications can occur anywhere in the polypeptide, including the peptide backbone, amino acid side chains, and the amino terminus or carboxyl terminus. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide can contain many types of modifications. Polypeptides can be branched, for example, as a result of ubiquitination, and polypeptides can be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides can result from natural post-translational processes or can be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, flavin covalent bond, heme moiety covalent bond, nucleotide or nucleotide derivative covalent bond, lipid or lipid derivative covalent bond, phosphatidylinositol Covalent bond, crosslinking, cyclization, disulfide bond formation, demethylation, covalent bridge formation, cysteine formation, pyroglutamic acid formation, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, Transfer of amino acids to proteins such as iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, arginylation R A mediated addition, and ubiquitination. (For example, PROTEINS-STRUCTURE AND MOLECULAR PROPERIES, 2nd edition, TE Creighton, WH Freeman and Company, New York (1993); POST-TRANSTRATIONAL COVALENT. Press, New York, pp. 1-12 (1983); Seifter et al., Meth. Enzymol. 182: 626-646 (1990); Rattan et al., Ann. NY Acad. Sci. 663: 48-62 (1992) checking).

(Functional activity)
“Polypeptide having functional activity” refers to a polypeptide that may exhibit one or more known functional activities associated with a full-length, proprotein, and / or mature form of a therapeutic protein. Such functional activities include biological activity, antigenicity [ability to bind (or compete with polypeptide for binding) to anti-polypeptide antibodies], immunogenicity (bind to a specific polypeptide of the invention). Ability to generate antibodies), the ability to form multimers with the polypeptides of the invention, and the ability to bind to receptors or ligands for the polypeptides include, but are not limited to.

  A “polypeptide with biological activity” is an activity similar to that of a therapeutic protein of the present invention, as measured in a particular biological assay, with or without dose dependence, A polypeptide (including a mature form) that exhibits an activity that is not necessarily identical to the activity. Where dose dependency exists, the dose dependency need not be the same as that of the polypeptide, but rather is substantially equivalent to the dose dependency in a given activity when compared to a polypeptide of the invention. (Ie, the candidate polypeptide has a higher activity compared to the polypeptide of the invention, or an activity of about 1/25 or less, preferably 1/10 or less, and most preferably about 1/3 or less. Showing).

  In preferred embodiments, the albumin fusion proteins of the invention have at least one biological and / or therapeutic activity associated with a therapeutic protein moiety (or fragment or variant thereof) when not fused to albumin.

  The albumin fusion proteins of the invention can be assayed for functional activity (eg, biological activity) using assays known in the art and those described herein or routinely modified. Can be done. In addition, those skilled in the art will use the assays referred to in the corresponding column of Table 1 (eg, column 3 of Table 1) to identify fragments of therapeutic proteins corresponding to the therapeutic protein portion of the albumin fusion protein for activity. It can be routinely assayed. In addition, one of ordinary skill in the art can use an assay known in the art and / or an albumin protein corresponding to the albumin protein portion of an albumin fusion protein for activity using the assays described in the Examples section below. Fragments can be routinely assayed.

  For example, in one embodiment in the art, an albumin fusion protein is assayed for the ability to bind to a therapeutic protein or to compete with the therapeutic protein for binding to an anti-therapeutic polypeptide antibody and / or anti-albumin antibody. A variety of known immunoassays can be used. Such assays include radioimmunoassays, ELISAs (enzyme-linked immunosorbent assays), “sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (eg, colloidal gold, enzymes or Radioisotope labeling), Western blots, precipitation reactions, agglutination assays (eg, gel aggregation assays, hemagglutination assays), complement binding assays, immunofluorescence assays, protein A assays, immunoelectrophoresis assays, and the like Competitive assay systems using technology and non-competitive assay systems include, but are not limited to. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detection of binding in an immunoassay and are within the scope of the present invention.

  In preferred embodiments, where a therapeutic protein binding partner (eg, receptor or ligand) is identified, it is well known in the art such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. By means, binding to a binding partner by an albumin fusion protein comprising that therapeutic protein as the therapeutic protein portion of the fusion can be assayed. See generally, Physicky et al., Microbiol. Rev. 59: 94-123 (1995). In another embodiment, the ability of the albumin fusion protein to correlate with a substrate of a therapeutic polypeptide corresponding to the therapeutic protein portion of the fusion is routinely determined using techniques known in the art. Can be assayed.

  In alternative embodiments, where the ability of the albumin fusion protein to multimerize is being assessed, association of the multimer with other components can be achieved, for example, by means well known in the art (eg, reduced and non-reduced). Gel chromatography, protein affinity chromatography, and affinity blotting). See generally Physicky et al. (Supra).

  In preferred embodiments, an albumin fusion protein comprising all or part of an antibody that binds to a therapeutic protein relates to an antibody that binds to the therapeutic protein (or fragment or variant thereof) when not fused to albumin. Have at least one biological and / or therapeutic activity (eg, specifically binding to a polypeptide or epitope). In other preferred embodiments, the biological and / or therapeutic activity of an albumin fusion protein comprising all or part of an antibody that binds to a therapeutic protein is specifically bound by an antibody that binds to the therapeutic protein. One or more inhibition (ie, antagonism) or activation (ie, agonistic action) of the biological and / or therapeutic activity associated with the polypeptide.

  Albumin fusion proteins comprising at least one fragment or variant of an antibody that binds to a therapeutic protein can be characterized in a variety of ways. In particular, an albumin fusion protein comprising at least one fragment or variant of an antibody that binds to a therapeutic protein is the same that is specifically bound by an antibody that binds to the therapeutic protein corresponding to the therapeutic protein portion of the albumin fusion protein. The ability to specifically bind to an antigen can be assayed using the techniques described herein, or routinely modifying techniques known in the art.

  An assay for the ability of an albumin fusion protein (eg, including at least one fragment or variant of an antibody that binds to a therapeutic protein) to bind (specifically) to a particular protein or epitope is performed in solution (eg, Houghten, Bio / Techniques 13: 412-421 (1992)), on beads (eg, Lam, Nature 354: 82-84 (1991)), on chip (eg, Fodor, Nature 364: 555-556 (1993)). )), On bacteria (eg, US Pat. No. 5,223,409), on spores (eg, US Pat. Nos. 5,571,698; 5,403,484; and 5,223,409), plasmids (E.g., Cull et al., Proc. Natl. cad.Sci.USA 89: 1865-1869 (1992)) or on phage (eg, Scott and Smith, Science 249: 386-390 (1990); Devlin, Science 249: 404-406 (1990); Cwirla et al. Proc. Natl. Acad. Sci. USA 87: 6378-6382 (1990); and Felici, J. Mol. Biol. 222: 301-310 (1991)) (each of these references is in its entirety, Which is incorporated herein by reference). Albumin fusion proteins comprising at least one fragment or variant of a therapeutic antibody are also known for their specificity and affinity for a particular protein or epitope as described herein or known in the art. Other methods can be used or routinely modified and assayed.

  An albumin fusion protein comprising at least one fragment or variant of an antibody that binds to a therapeutic protein may comprise other antigens (eg, a therapeutic protein (or fragment or fragment thereof) corresponding to the therapeutic protein portion of the albumin fusion protein of the invention. Cross-reactivity with molecules that have sequence / structure conservation with molecules that are specifically bound by antibodies that bind to variants) can be assayed by any method known in the art.

  Immunoassays that can be used to analyze (immunospecific) binding and cross-reactivity include, but are not limited to, Western blots, radioimmunoassays, ELISAs (enzyme-linked immunosorbent assays), “sandwich” immunoassays, Competitive assay systems and non-competitive using techniques such as immunoprecipitation assay, sedimentation reaction, gel diffusion sedimentation reaction, immunodiffusion assay, aggregation assay, complement binding assay, immunoradiometric assay, fluorescent immunoassay, and protein A immunoassay Examples include, but are not limited to, assay systems. Such assays are conventional and well known in the art (see, eg, Ausubel et al., 1994, Current Protocols in Molecular Biology, Vol. 1, which is incorporated herein by reference in its entirety. See John Wiley & Sons, Inc., New York). Exemplary immunoassays are briefly described below (although these are not intended to be limiting).

  Immunoprecipitation protocols generally involve RIPA buffer (1% NP-40 or Triton X-100, 1% deoxychol) supplemented with protein phosphatase inhibitors and / or protease inhibitors (eg, EDTA, PMSF, aprotinin, sodium vanadate). Lysing a population of cells in a lysis buffer such as sodium acid, 0.1% SDS, 0.15M NaCl, 0.01M sodium phosphate (pH 7.2), 1% Trasylol), Adding an albumin fusion protein (eg, comprising at least one fragment or variant of an antibody that binds to a therapeutic protein) to the cell lysate, incubating at 40 ° C. for a period of time (eg, 1-4 hours); For example, anti-albumin anti Adding Sepharose beads bound to to cell lysate, incubating at 40 ° C. for about 1 hour or more, washing beads in lysis buffer, and resuspending beads in SDS / sample buffer Process. The ability of an albumin fusion protein to immunoprecipitate a particular antigen can be assayed, for example, by Western blot analysis. One skilled in the art will be concerned with parameters that can be modified to increase the binding of the albumin fusion protein to the antigen and to reduce the background (e.g., preclean the cell lysate using Sepharose beads). There can be knowledge. For further discussion on immunoprecipitation protocols, see, eg, Ausubel et al., 1994, Current Protocols in Molecular Biology, Volume 1, John Wiley & Sons, Inc. , New York, 10.16.1.

Western blot analysis generally involves preparing a protein sample, electrophoresis of the protein sample on a polyacrylamide gel (eg, 8-20% SDS-PAGE depending on the molecular weight of the antigen), and the protein sample on a polyacrylamide gel. Transfer to a membrane (eg nitrocellulose, PVDF or nylon), block the membrane in a blocking solution (eg PBS containing 3% BSA or non-fat milk), wash the membrane (eg PBS- Washing in Tween 20), applying the albumin fusion protein of the present invention (diluted with blocking buffer) to the membrane, washing the membrane in washing buffer, diluted with blocking buffer, enzyme Substrate (for example, , Applying a horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., ^{32 P} or ^{125 I)} in the conjugated secondary antibody (which albumin fusion proteins (e.g., anti-human serum albumin antibody) recognizes) Washing the membrane in a wash buffer and detecting the presence of the antigen. Those skilled in the art are familiar with parameters that can be modified to increase the detected signal and reduce background noise. For further discussion on Western blot protocols, see, eg, Ausubel et al. (Eds.), 1994, Current Protocols in Molecular Biology, Volume 1, John Wiley & Sons, Inc. , New York (10.8.1).

  An ELISA is conjugated to a detectable compound such as preparing an antigen, coating the wells of a 96-well microtiter plate with the antigen, an enzyme substrate (eg, horseradish peroxidase or alkaline phosphatase). Adding an albumin fusion protein of the invention (eg, comprising at least a fragment or variant of an antibody that binds to a therapeutic protein) to the well and incubating for a period of time, unbound albumin fusion protein or unspecifically bound albumin Washing the fusion protein, and detecting the presence of albumin fusion protein specifically bound to the antigen that coats the well. In ELISA, the antibody of interest need not be conjugated to a detectable compound; instead, a second antibody conjugated to a detectable compound (recognizing an albumin fusion protein) is added to the well. Can be done. Further, instead of coating the well with the antigen, the albumin fusion protein can be coated to the well. In this case, the detectable molecule can be an antigen conjugated to a detectable compound (enzyme substrate such as horseradish peroxidase or alkaline phosphatase. Those skilled in the art will increase the signal detected. You may have knowledge about the parameters that can be modified and other variations of ELISA known in the art For further discussion on ELISA, see, eg, Ausubel et al., 1994, Current Protocols in Molecular Biology, Volume 1, John. See Wiley & Sons, Inc., New York, 11.2.1.

The binding affinity of an albumin fusion protein for a protein, antigen, or epitope and the off-rate of the albumin fusion protein-protein / antigen / epitope interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay, which comprises a labeled antigen (eg, ³ H or ¹²⁵ I) and an albumin fusion protein of the invention in the presence of increasing amounts of unlabeled antigen. And detection of antibodies bound to the labeled antigen.
The affinity of an albumin fusion protein for a particular protein, antigen or epitope, and binding off-rate can be determined from data by Scatchard plot analysis. Competition of the albumin fusion protein with a second protein that binds to the same protein, antigen or epitope can also be determined using radioimmunoassay. In this case, the protein, antigen, or epitope is incubated with an albumin fusion protein conjugated to a labeled compound (eg, ³ H or ¹²⁵ I) in the presence of increasing amounts of unlabeled second antibody.

  In a preferred embodiment, BIAcore kinetic analysis is used to determine the on and off rates of binding of an albumin fusion protein of the invention to a protein, antigen or epitope. BIAcore kinetic analysis is performed on albumin fusion proteins, or albumin from a chip comprising a specific polypeptide, antigen, or epitope, respectively, and a specific polypeptide, antigen, or epitope, or albumin fusion protein immobilized on the surface. Analyzing the dissociation of the fusion protein, or a specific polypeptide, antigen, or epitope.

An antibody that binds to a therapeutic protein corresponding to a therapeutic protein portion of an albumin fusion protein is also described or specified with respect to binding affinity for a given protein or antigen, preferably an antigen to which their alternation specifically binds. Can be done. Preferred binding affinities are less than 5 × 10 ⁻² M, less than 10 ⁻² M, less than 5 × 10 ⁻³ M, less than 10 ⁻³ M, less than 5 × 10 ⁻⁴ M, and less than 10 ⁻⁴ M. Affinities with a constant or Kd are mentioned. More preferable binding affinity is less than 5 × 10 ⁻⁵ M, less than 10 ⁻⁵ M, less than 5 × 10 ⁻⁶ M, less than 10 ⁻⁶ M, less than 5 × 10 ⁻⁷ M, less than 10 ⁻⁷ M, Affinities having a dissociation constant, ie Kd, of less than 5 × 10 ⁻⁸ M and less than 10 ⁻⁸ M. More preferably, the binding affinity is less than 5 × 10 ⁻⁹ M, less than 10 ⁻⁹ M, less than 5 × 10 ⁻¹⁰ M, less than 10 ⁻¹⁰ M, less than 5 × 10 ⁻¹¹ M, less than 10 ⁻¹¹ M. Less than 5 × 10 ⁻¹² M, less than 10 ⁻¹² M, less than 5 × 10 ⁻¹³ M, less than 10 ⁻¹³ M, less than 5 × 10 ⁻¹⁴ M, less than 10 ⁻¹⁴ M, less than 5 × 10 ⁻¹⁵ M Alternatively, an affinity having a dissociation constant, that is, a Kd of less than 10 ⁻¹⁵ M can be mentioned. In a preferred embodiment, the albumin fusion protein comprising at least a fragment or variant of an antibody that binds to a therapeutic protein has a predetermined similarity that is similar to the affinity of the corresponding antibody (not fused to albumin) that binds to the therapeutic protein. Having an affinity for a protein or epitope, considering the value of the albumin fusion protein (including at least a fragment or variant of an antibody that binds to a therapeutic protein) and its corresponding antibody value. In addition, the assays described herein (see Examples and Table 1), otherwise known in the art, show that albumin fusion proteins and fragments, variants and derivatives thereof are albumin fusions. Conventionally applied to measure the ability of a protein to induce biological activity and / or therapeutic activity (either in vitro or in vivo) associated with either a therapeutic protein portion and / or an albumin portion. obtain. Other methods are known to those skilled in the art and are within the scope of the present invention.

(albumin)
As mentioned above, the albumin fusion protein of the present invention comprises at least a fragment or variant of a therapeutic protein and at least a fragment or variant of human serum albumin, which are bound to each other, preferably by gene fusion. Has been.

  Further embodiments include at least a fragment or variant of a therapeutic protein and at least a fragment or variant of human serum albumin, which are linked to each other by chemical bonds.

  The terms human serum albumin (HSA) and human albumin (HA) are used interchangeably herein. The terms “albumin” and “serum albumin” are broader and include human serum albumin (and fragments and variants thereof) and albumin from other species (and fragments and variants thereof).

  As used herein, “albumin” refers to a protein or amino acid sequence of albumin or a fragment or variant of albumin that has one or more functional activities (eg, biological activity) of albumin. Collectively. In particular, “albumin” refers to human albumin or fragments thereof (eg EP 201 239, EP 322 094, WO 97/24445, WO 95/23857), in particular human albumin as shown in FIG. 1 and SEQ ID NO: 1038. Or other analogs or variants of these molecules or fragments.

  In a preferred embodiment, the human serum albumin used in the albumin fusion protein of the invention comprises one or both of the following sets of point mutations relating to SEQ ID NO: 1038: Leu-407 → Ala, Leu-408 → Val, Val-409 → Ala, and Arg-410 → Ala; or Arg-410 → A, Lys-413 → Gln, and Lys-414 → Gln (eg, incorporated herein by reference in their entirety, (See International Publication No. WO 95/23857). In an even more preferred embodiment, the albumin fusion protein of the invention comprising one or both of the above point mutation sets has improved stability / resistance to yeast Yap3p proteolytic cleavage, thereby Increased production of recombinant albumin fusion proteins expressed in cells is possible.

As used herein, an albumin moiety sufficient to extend the therapeutic activity or longevity of a therapeutic protein is sufficient in length or structure to stabilize or prolong the therapeutic activity of the protein. As a result, it refers to an albumin moiety in which the lifetime of the therapeutic protein portion of the albumin fusion protein is extended or extended compared to the lifetime in the unfused state. The albumin portion of the albumin fusion protein can include the full length of the HA sequence as described above, or can include one or more fragments thereof that can stabilize or extend its therapeutic activity. Such a fragment can be 10 or more amino acids in length, or can comprise about 15, 20, 25, 30 or more consecutive amino acids from the HA sequence, or HA. Part or all of a particular domain.
For example, one or more fragments of HA that span the first two immunoglobulin-like domains can be used. In a preferred embodiment, the HA fragment is the mature form of HA.

  The albumin portion of the albumin fusion protein of the present invention can be a variant of normal HA. The therapeutic protein portion of an albumin fusion protein of the invention can also be a variant of a therapeutic protein as described herein. The term “variant” includes insertions, deletions, and substitutions, either conservative or non-conservative, where such changes are useful for the oncotic useful ligand binding properties. And one or more of the non-immunogenic properties, nor the active site or domain conferring the therapeutic activity of the therapeutic protein is substantially unchanged.

  Specifically, the albumin fusion proteins of the present invention are naturally occurring polymorphic variants of human albumin, and fragments of human albumin (eg, fragments disclosed in EP 322 094 (ie, HA (Pn)) Where n is 369-419))). The albumin can be derived from any vertebrate, especially any mammal, such as humans, cows, sheep, or pigs. Non-mammalian albumin includes, but is not limited to, hens and salmon. The albumin portion of the albumin fusion protein can be from a different animal than the therapeutic protein portion.

  Generally speaking, a fragment or variant of HA is at least 100 amino acids long, preferably at least 150 amino acids long. This HA variant comprises at least one whole domain of HA, such as domain 1 (amino acids 1 to 194 of SEQ ID NO: 1038), domain 2 (amino acids 195 to 387 of SEQ ID NO: 1038), domain 3 (amino acids of SEQ ID NO: 1038) 388-585), domains 1 and 2 (1 to 387 of SEQ ID NO: 1038), domains 2 and 3 (195 to 585 of SEQ ID NO: 1038), or domains 1 and 3 (amino acids 1 to 194 of SEQ ID NO: 1038 and SEQ ID NO: 1038 amino acids 388-585) or may comprise these. Each domain itself is composed of two homologous subdomains: 1-105, 120-194, 195-291, 316-387, 388-491, and 512-585, a flexible intersubdomain linker The region includes residues Lys106-Glu119, Glu292-Val315, and Glu492-Ala511.

  Preferably, the albumin portion of the albumin fusion protein of the invention comprises at least one subdomain or domain of HA or a conservative variant thereof. If the fusion is based on a subdomain, some or all of the adjacent linkers are preferably used to attach to a therapeutic protein moiety.

(Antibodies that specifically bind to a therapeutic protein are also therapeutic proteins)
The invention also encompasses albumin fusion proteins comprising at least a fragment or variant of an antibody that specifically binds to a therapeutic protein disclosed in Table 1. The term “therapeutic protein” is specifically contemplated to include antibodies (eg, listed in column I of Table 1) and fragments and variants thereof that bind to the therapeutic protein. Thus, an albumin fusion protein of the invention can comprise at least a fragment or variant of a therapeutic protein and / or at least a fragment or variant of an antibody that binds to the therapeutic protein.

(Antibody structure and background)
The basic antibody structural unit is known to contain a tetramer. Each tetramer is composed of two identical polypeptide chain pairs, each pair having one “light chain” (about 25 kDa) and one “heavy chain” (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region f of about 100 to 110 amino acids or more that is mainly responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector functions. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. See generally, Fundamental Immunology, Chapters 3-5 (Paul, W. Ed., 4th edition, Raven Press, NY (1998), which is incorporated by reference in its entirety for all purposes). The variable region of each light / heavy chain pair forms the antibody binding site.

  Thus, an intact IgG antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are the same.

  All of these chains exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions (also called complementarity determining regions or CDRs). This CDR region is generally the portion of the antibody that contacts the antigen to determine its specificity. The CDRs from each pair of heavy and light chains are aligned by the framework region, and are thus capable of binding to a specific epitope. From the N-terminus to the C-terminus, both the light chain variable region and the heavy chain variable region contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. These variable regions are linked to the heavy chain constant region or the light chain constant region. The assignment of amino acids to each domain was determined by Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk. Biol. 196: 901-917 (1987); Chothia et al., Nature 342: 878-883 (1989).

  As used herein, “antibody” refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules (ie, molecules that contain an antigen binding site that specifically binds an antigen (eg, Molecule)) containing one or more CDR regions of an antibody. Antibodies that can correspond to the therapeutic protein portion of an albumin fusion protein include monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies or chimeric antibodies, single chain antibodies (eg, single chain Fvs), Fab fragments, F ( ab ') fragments, fragments produced by Fab expression libraries, anti-idiotype (anti-Id) antibodies (including anti-Id antibodies specific for antibodies of the invention), and any epitope binding fragment described above (Eg, but not limited to, a VH domain, a VL domain, or one or more CDR regions).

(Antibodies that bind to therapeutic proteins)
The invention encompasses albumin fusion proteins comprising at least a fragment or variant of an antibody that binds to a therapeutic protein (eg, disclosed in Table 1), or a fragment or variant thereof.

  The antibody that binds to the therapeutic protein (or fragment or variant thereof) can be from any animal origin, including birds and mammals. Preferably, the antibody is a human, murine (eg, mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken antibody. More preferably, the antibody is a human antibody. As used herein, a “human” antibody includes an antibody having the amino acid sequence of a human immunoglobulin, and an antibody isolated from a human immunoglobulin library, and a gene that produces a human antibody. It includes antibodies isolated from engineered xenomic mice or other organisms.

  Antibody molecules that bind to a therapeutic protein and can correspond to the therapeutic protein portion of an albumin fusion protein of the invention can be any type of immunoglobulin molecule (eg, IgG, IgE, IgM, IgD, IgA, and IgY). , Any class (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or any subclass. In a preferred embodiment, the antibody molecule that can bind to the therapeutic protein and correspond to the therapeutic protein portion of an albumin fusion protein is IgG1. In another preferred embodiment, the antibody molecule that can bind to the therapeutic protein and correspond to the therapeutic protein portion of an albumin fusion protein is IgG2. In another preferred embodiment, the antibody molecule that can bind to the therapeutic protein and correspond to the therapeutic protein portion of an albumin fusion protein is IgG4.

Most preferably, the antibody that can bind to the therapeutic protein and correspond to the therapeutic protein portion of the albumin fusion protein is a human antigen-binding fragment of the invention, including Fab, Fab ′ and F (ab ′) ₂ , Fd, single chain Fvs (scFv), single chain antibodies, disulfide bond Fvs (sdFv), and fragments containing either VL or VH domains. Antigen-binding antibody fragments, including single chain antibodies, can include variable regions, alone or in combination with all or part of the following: hinge region, CH1 domain, CH2 domain and CH3 domain.

  An antibody that can bind to a therapeutic protein and correspond to a therapeutic protein portion of an albumin fusion protein can be monospecific, bispecific, trispecific or more specific. Multispecific antibodies can be specific for different epitopes of the therapeutic protein, or can be specific for both the therapeutic protein and a heterologous epitope (eg, a heterologous polypeptide or solid support material). For example, PCT Publication WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt et al. Immunol. 147: 60-69 (1991); U.S. Pat. Nos. 4,474,893, 4,714,681, 4,925,648, 5,573,920, 5, 601,819; Kostelny et al., J. MoI. Immunol. 148: 1547-1553 (1992).

  An antibody that binds to a therapeutic protein (or fragment or variant thereof) can be bispecific or bifunctional, which means that the antibody has two different heavy / light chain pairs and two different binding sites. Is an artificial hybrid antibody. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or binding of Fab 'fragments. See, for example, Songsivirai and Lachmann, Clin. Exp. Immunol. 79: 315-321 (1990), Kostelny et al., J. Biol. Immunol. 148: 1547-1553 (1992). In addition, bispecific antibodies can be identified as “diabodies” (Holligner et al., “Diabodies': small bivalent and bispecific antigenic fragments” PNAS USA 90: 6444-6448 (1993)) or “Jerin et al.”. Bisific single chain moles (Janusins) target cytotoxic lymphocytos on HIV infected cells, EMBO J 10: 3653-3659 (1991) and Traunecker et al "Janusnewne gents "Int J Cancer Suppl 7: can be found as 51-52 (1992)).

  The invention also provides albumin fusion proteins comprising antibody fragments or variants (including derivatives) as described herein or known in the art. Standard techniques known to those skilled in the art can be used to introduce mutations into the nucleotide sequences encoding the molecules of the invention (eg, site-directed mutagenesis and PCR-mediated mutagenesis resulting in amino acid substitutions). ). Preferably, this variant (including derivatives) has less than 50 amino acid substitutions, less than 40 amino acid substitutions compared to a reference VH domain, VHCDR1, VHCDR2, VHCDR3, VL domain, VLCDR1, VLCDR2, or VLCDR3 Less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 15 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, Encodes less than 3 amino acid substitutions, or less than 2 amino acid substitutions. In certain embodiments, the variants encode VHCDR3 substitutions. In preferred embodiments, the variants have conservative amino acid substitutions at one or more putative non-essential amino acid residues.

  Antibodies that can bind to a therapeutic protein and correspond to a therapeutic protein portion of an albumin fusion protein can be described or specified with respect to an epitope or portion of the therapeutic protein that they recognize or specifically bind. Antibodies that specifically bind to a therapeutic protein or to a specific epitope of a therapeutic protein can also be eliminated. Thus, the present invention includes antibodies that specifically bind and allow the elimination of therapeutic proteins. In preferred embodiments, an albumin fusion protein comprising at least a fragment or variant of an antibody that binds to a therapeutic protein binds to the same epitope as a non-fusion fragment or variant of the antibody itself.

Antibodies that can bind to a therapeutic protein and correspond to the therapeutic protein portion of an albumin fusion protein can also be described or specified for their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homolog of a therapeutic protein are included.
At least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55% and at least 50% identity to the therapeutic protein Antibodies that bind polypeptides having (as calculated using methods known in the art and methods described herein) are also included in the invention. In certain embodiments, antibodies that can bind to a therapeutic protein and correspond to a therapeutic protein portion of an albumin fusion protein cross a mouse homologue, rat homologue and / or rabbit homologue of human protein and their corresponding epitopes. react. <95%, <90%, <85%, <80%, <75%, <70%, <65%, <60%, <55% and <50% sequence identity to therapeutic protein ( Antibodies that do not bind polypeptides with (as calculated using methods known in the art and methods described herein) are also included in the invention. In certain embodiments, the cross-reactivity described above is any single specific antigenic polypeptide or immunogenic polypeptide disclosed herein, or a specific antigenic polypeptide thereof. And / or a combination of two, three, four, five or more of the immunogenic polypeptides. In a preferred embodiment, an albumin fusion protein comprising at least a fragment or variant of an antibody that binds to a therapeutic protein is similar or substantially identical crossing compared to a fragment or variant of the particular antibody itself. Has reactivity.

Further included in the present invention is a polypeptide encoded by a polynucleotide that hybridizes under stringent hybridization conditions (as described herein) to a polynucleotide encoding a therapeutic protein. It is an antibody that binds. Antibodies that can bind to therapeutic proteins and correspond to the therapeutic protein portion of an albumin fusion protein can also be described or specified for their binding affinity to the polypeptides of the invention. Preferred binding affinities are less than 5 × 10 ⁻² M, less than 10 ⁻² M, less than 5 × 10 ⁻³ M, less than 10 ⁻³ M, less than 5 × 10 ⁻⁴ M, and less than 10 ⁻⁴ M. Affinities with a constant or Kd are mentioned. More preferable binding affinity is less than 5 × 10 ⁻⁵ M, less than 10 ⁻⁵ M, less than 5 × 10 ⁻⁶ M, less than 10 ⁻⁶ M, less than 5 × 10 ⁻⁷ M, less than 10 ⁻⁷ M, Affinities having a dissociation constant or Kd of less than 5 × 10 ⁻⁸ M or less than 10 ⁻⁸ M are mentioned. More preferably, the binding affinity is less than 5 × 10 ⁻⁹ M, less than 10 ⁻⁹ M, less than 5 × 10 ⁻¹⁰ M, less than 10 ⁻¹⁰ M, less than 5 × 10 ⁻¹¹ M, less than 10 ⁻¹¹ M. Less than 5 × 10 ⁻¹² M, less than 10 ⁻¹² M, less than 5 × 10 ⁻¹³ M, less than 10 ⁻¹³ M, less than 5 × 10 ⁻¹⁴ M, less than 10 ⁻¹⁴ M, less than 5 × 10 ⁻¹⁵ M Alternatively, an affinity having a dissociation constant, that is, a Kd of less than 10 ⁻¹⁵ M can be mentioned. In a preferred embodiment, the albumin fusion protein comprising at least a fragment or variant of an antibody that binds to a therapeutic protein has a predetermined similarity that is similar to the affinity of the corresponding antibody (not fused to albumin) that binds to the therapeutic protein. Having an affinity for a protein or epitope, considering the value of the albumin fusion protein (including at least a fragment or variant of an antibody that binds to a therapeutic protein) and its corresponding antibody value.

  The invention also relates to the binding of an antibody to an epitope of a therapeutic protein, as determined by any method known in the art for determining competitive binding (eg, an immunoassay described herein). An antibody that competitively inhibits is provided. In preferred embodiments, the antibody is at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%, competitive for binding to that epitope. To inhibit. In preferred embodiments, an albumin fusion protein comprising at least a fragment or variant of an antibody that binds to a therapeutic protein competitively inhibits the binding of a second antibody to an epitope of the therapeutic protein. In other preferred embodiments, the albumin fusion protein comprising at least a fragment or variant of an antibody that binds to a therapeutic protein has a second antibody binding to an epitope of the therapeutic protein of at least 95%, at least 90%, Inhibit competitively by at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%.

  An antibody that binds to a therapeutic protein and can correspond to a therapeutic protein portion of an albumin fusion protein of the invention can act as an agonist or antagonist of the therapeutic protein. For example, the invention includes antibodies that partially or completely disrupt receptor / ligand interactions with the polypeptides of the invention. The present invention has features of both receptor-specific antibodies and ligand-specific antibodies. The present invention also features receptor-specific antibodies that do not interfere with ligand binding but interfere with receptor activation. Receptor activation (ie, signal transduction) can be determined by techniques described herein, otherwise techniques known in the art. For example, receptor activation can be determined by detecting the substrate by receptor phosphorylation (eg, tyrosine or serine / threonine), or immunoprecipitation followed by Western blot analysis (eg, as described above). . In certain embodiments, the ligand or receptor activity in the absence of the antibody is at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or Antibodies that inhibit at least 50% are provided. In a preferred embodiment, an albumin fusion protein comprising at least a fragment or variant of an antibody that binds to a therapeutic protein prevents ligand binding and compares to an unfused fragment or variant of an antibody that binds to the therapeutic protein. Have similar or substantially similar characteristics with respect to prevention of receptor activation.

  The present invention also specifically recognizes receptor-specific antibodies that prevent both ligand binding and receptor activation, and receptor-ligand complexes, and preferably unbound and unbound ligands specifically. It has the characteristics of a receptor-specific antibody that is not recognized. Similarly, the present invention provides a neutralizing antibody that binds to a ligand and prevents binding of that ligand to a receptor, and binds to a ligand, thereby preventing receptor activation but preventing the ligand from binding the receptor. Contains no antibodies. Furthermore, the present invention includes antibodies that activate the receptor. These antibodies can act as receptor agonists, ie, enhance either all or a subset of the biological activity of ligand-mediated receptor activation, eg, by inducing dimerization of the receptor. Or can be activated. The antibody can be specified as an agonist, antagonist or inverse agonist for a biological activity including the specific biological activity of a therapeutic protein disclosed herein (eg, disclosed in Table 1). . The antibody agonist can be produced using a method known in the art. See, for example, PCT Publication WO 96/40281; US Pat. No. 5,811,097; Deng et al., Blood 92 (6): 1981-1988 (1998); Chen et al., Cancer Res. 58 (16): 3668-3678 (1998); Harrop et al., J. Biol. Immunol. 161 (4): 1786-1794 (1998); Zhu et al., Cancer Res: 58 (15): 3209-3214 (1998); Yoon et al., J. Biol. Immunol. 160 (7): 3170-3179 (1998); Prat et al., J. Biol. Cell. Sci. 111 (Pt2): 237-247 (1998); Pitald et al., J. Biol. Immunol. Methods 205 (2): 177-190 (1997); Liautard et al., Cytokine 9 (4): 233-241 (1997); Carlson et al., J. Biol. Biol. Chem. 272 (17): 11295-11301 (1997); Taryman et al., Neuron 14 (4): 755-762 (1995); Muller et al., Structure 6 (9): 1153-1167 (1998); Bartunek et al., Cytokine 8 ( 1): 14-20 (1996) (all of the above references are incorporated herein by reference in their entirety). In preferred embodiments, an albumin fusion protein comprising at least a fragment or variant of an antibody that binds to a therapeutic protein is similar or substantially comparable to an unfused fragment or variant of an antibody that binds to the therapeutic protein. Have the same agonistic or antagonistic properties.

  Antibodies that can bind to a therapeutic protein and correspond to the therapeutic protein portion of an albumin fusion protein of the invention can be used, for example, to purify, detect, and target the therapeutic protein. These include both in vitro and in vivo diagnostic and therapeutic methods. For example, the antibody has utility in an immunoassay for qualitatively and quantitatively measuring the level of a therapeutic protein in a biological sample. See, for example, Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd Edition, 1988), which is hereby incorporated by reference in its entirety. Similarly, an albumin fusion protein comprising at least a fragment or variant of an antibody that binds to a therapeutic protein can be used, for example, to purify, detect, and target the therapeutic protein. These include both in vitro and in vivo diagnostic and therapeutic methods.

  Antibodies that can bind to a therapeutic protein and correspond to the therapeutic protein portion of an albumin fusion protein include modified derivatives (ie, by covalent attachment of any type of molecule to the antibody). For example, without limitation, the antibody derivative may be, for example, glycosylated, acetylated, PEGylated, phosphorylated, amidated, derivatized with a known protecting / blocking group, proteolytic cleavage, cellular ligand or other Antibodies that have been modified, such as by binding to a protein, are included. Any of a number of chemical modifications can be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, the derivative may contain one or more non-classical amino acids. The albumin fusion protein of the invention can also be modified as described above.

(Method of generating antibody binding to therapeutic protein)
Antibodies that bind to a therapeutic protein and can correspond to a therapeutic protein portion of an albumin fusion protein of the invention can be produced by any suitable method known in the art. Polyclonal antibodies against the antigen of interest can be produced by various procedures well known in the art. For example, therapeutic proteins can be administered to various host animals (including but not limited to rabbits, mice, rats, etc.) to induce the production of sera containing polyclonal antibodies specific for that antigen. Various adjuvants can be used to increase the immunological response, depending on the host species, and surfactants such as Freund (complete and incomplete), mineral gels such as aluminum hydroxide, lysolecithin , Pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and corynebacterium parvum, It is not limited. Such adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or combinations thereof. For example, monoclonal antibodies can be produced using the following hybridoma techniques known in the art, for example, Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd edition, 1988); Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY 1981), which is incorporated by reference in its entirety. As used herein, the term “monoclonal antibody” is not limited to antibodies produced through hybridoma technology.
The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not from the method by which it is produced.

  Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art. In a non-limiting example, the mouse uses a therapeutic protein or fragment or variant thereof, an albumin fusion protein, or a cell expressing such therapeutic protein or fragment or variant or albumin fusion protein, Can be immunized. Once an immune response is detected (eg, antibodies specific for the antigen are detected in the mouse serum), the mouse spleen is collected and splenocytes are isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells (eg, cells from cell line SP20 available from ATCC). Hybridomas are selected and cloned by limiting dilution. The hybridoma clones are then assayed by methods known in the art for cells secreting antibodies capable of binding to the polypeptides of the invention. Ascites fluid, which generally contains high levels of antibodies, can be produced by immunizing mice with positive hybridoma clones.

  Accordingly, the present invention provides a method for producing a monoclonal antibody and an antibody produced by a method comprising culturing a hybridoma cell that secretes the antibody, wherein preferably the hybridoma is a myeloma. By screening the hybridoma resulting from the fusion for hybridoma clones that secrete antibodies capable of binding to the polypeptide of the invention, and then fusing the cells with splenocytes isolated from a mouse immunized with the antigen of the invention Generated.

Another well known method for generating both polyclonal and monoclonal human B cell lines is transformation using Epstein Barr Virus (EBV).
Protocols for generating EBV-transformed B cell lines are generally known in the art, for example, Current Protocols in Immunology, Coligan et al., 1994, John Wiley & Sons, NY (this is incorporated by reference in its entirety) There are protocols outlined in Section 7.22 of (incorporated herein).
The source of B cells for transformation is generally human peripheral blood, although B cells for transformation also include other sources (lymph nodes, tonsils, spleen, tumor tissue, and infected tissue, (But not limited to). Tissue is generally made into a single cell suspension prior to EBV transformation. In addition, a step for physically removing or inactivating (eg, by treatment with cyclosporin A) T cells in the B cell-containing sample can be performed. This is because T cells derived from individuals who are seropositive for anti-EBV antibodies can suppress B cell immortalization by EBV.

  Generally, a sample containing human B cells is inoculated with EBV and cultured for 3-4 weeks. A typical EBV source is the culture supernatant of the B95-8 cell line (ATCC number VR-1492). Physical signs of EBV transformation can generally be observed at the end of this 3-4 week culture period. By phase contrast microscopy, transformed cells can appear large, transparent, hairy and tend to aggregate into tight cell clusters. Initially, EBV strains are generally polyclonal. However, during long cell culture periods, EBV strains can become monoclonal or polyclonal as a result of selective growth of specific B cell clones. Alternatively, a polyclonal EBV transformant can be subcloned (eg, by limiting dilution culture) or fused with an appropriate fusion partner and plated at limiting dilution to obtain a monoclonal B cell line. Also obtain a monoclonal B cell line. Suitable fusion partners for EBV transformed cell lines include mouse myeloma cell lines (eg SP2 / 0, X63-Ag8.653), heteromyeloma cell lines (human x mouse; eg SPAM-8, SBC) -H20, and CB-F7), and human cell lines (eg, GM 1500, SKO-0007, RPMI 8226, and KR-4). Accordingly, the present invention also provides a method for producing a human polyclonal antibody or human monoclonal antibody against the polypeptide of the present invention or a fragment thereof (including EBV transformation of human B cells).

Antibody fragments that recognize specific epitopes can be generated by known techniques.
For example, the Fab and F (ab ′) 2 fragments of the present invention can be obtained using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F (ab ′) 2 fragments). Can be produced by proteolytic cleavage of immunoglobulin molecules. The F (ab ′) 2 fragment contains the variable region, the light chain constant region and the CH1 domain of the heavy chain.

  For example, antibodies that bind to therapeutic proteins can also be produced using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles that carry the polynucleotide sequences that encode them. In certain embodiments, such phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library (eg, human or mouse). Phage expressing an antigen binding domain that binds to the antigen of interest can be selected or identified using the antigen (eg, using labeled antigen or antigen bound or captured to a solid surface or bead). Phages used in these methods are typically phage having Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either phage gene III protein or phage gene VIII protein. Filamentous phage containing fd and M13 binding domains expressed from. Examples of phage display methods that can be used to generate antibodies that bind to therapeutic proteins include the methods disclosed below: Brinkman et al. Immunol. Methods 182: 41-50 (1995); Ames et al., J. Biol. Immunol. Methods 184: 177-186 (1995); Kettleborough et al., Eur. J. et al. Immunol. 24: 952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57: 191-280 (1994); PCT application number PCT / GB91 / 01134; PCT publication WO 90 / WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and US Pat. No. 5,698,426; No. 5,403,484; No. 5,580,717; No. 5,427,908; No. 5,750,753; No. 5,821,047; No. 5 No. 5,571,698; No. 5,427,908; No. 5,516,6 No. 7; No. 5,780,225; No. 5,658,727; No. 5,733,743 and No. 5,969,108, each of which is The whole is incorporated by reference).

As described in the above references, after phage selection, the region encoding a phage-derived antibody is isolated to produce the entire antibody, including human antibodies, or any other desired antigen-binding fragment and And can be expressed in any desired host, including, for example, mammalian cells, insect cells, plant cells, yeast and bacteria, as described in detail below. For example, techniques for recombinantly producing Fab, Fab ′ and F (ab ′) 2 fragments can also be used using methods known in the art, and such methods are disclosed below. PCT Publication WO 92/22324; Mullinax et al., BioTechniques 12 (6): 864-869 (1992); and Sawai et al., AJRI 34: 26-34 (1995); and Better et al., Science 240: 1041-1043 ( (1998) (These references are incorporated by reference in their entirety).

  Examples of techniques that can be used to produce single chain Fvs and antibodies include US Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203: 46-88. (1991); Shu et al., PNAS 90: 7995-7999 (1993); and Skerra et al., Science 240: 1038-1040 (1988). For some uses, including in vivo use of antibodies in humans and in vitro detection assays, the use of chimeric, humanized or human antibodies may be preferred. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species (eg, antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region). Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, Science 229: 1202 (1985); Oi et al., BioTechniques 4: 214 (1986); Gillies et al. (1989) J. MoI. Immunol. Methods 125: 191-202; US Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which are hereby incorporated by reference in their entirety. Incorporated in). A humanized antibody is an antibody molecule derived from a non-human species antibody that binds to a desired antigen having one or more complementarity determining regions (CDRs) from a non-human species and a framework region from a human immunoglobulin molecule. . Often, framework residues within the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter (preferably improve) antigen binding. These framework substitutions are identified by methods well known in the art, such as modeling CDR and framework residue interactions to identify framework residues important for antigen binding, and at specific positions. By sequence comparison to identify unusual framework residues. (See, eg, Queen et al., US Pat. No. 5,585,089; Riechmann et al., Nature 332: 323 (1988), which are hereby incorporated by reference in their entirety). Antibodies can be humanized using various techniques known in the art including, for example: CDR-graft (European Patent No. 239,400; PCT Publication WO 91/09967; US Patent No. 5,225) No. 539; Nos. 5,530,101 and 5,585,089), veneering or resurfacing (European Patent No. 592,106; No. 519,596); Padlan, Molecular Immunology 28 (4/5): 489-498 (1991); Studnikka et al., Protein Engineering 7 (6): 805-814 (1994); Roguska et al., PNAS 91: 969-973 (1994). Chain shuffling (c ain shuffling) (US Pat. No. 5,565,332).

  Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be produced by various methods known in the art, including the phage display method described above using an antibody library derived from human immunoglobulin sequences. U.S. Pat. Nos. 4,444,887, 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96 / See also 33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety.

Human antibodies can also be produced using transgenic mice that are unable to express functional endogenous immunoglobulins but are capable of expressing human immunoglobulin genes. For example, a complex of a human heavy chain immunoglobulin gene and a human light chain immunoglobulin gene can be introduced into mouse embryonic stem cells randomly or by homologous recombination. Alternatively, human variable regions, constant regions and diversity regions can be introduced into mouse embryonic stem cells in addition to human heavy chain genes and human light chain genes. The mouse heavy and light chain immunoglobulin genes can be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. Modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring that express human antibodies. Transgenic mice are immunized in the normal fashion with a selected antigen (eg, all or part of a polypeptide of the invention). Monoclonal antibodies against the antigen can be obtained from immunized transgenic mice using conventional hybridoma technology. Human immunoglobulin transgenes retained in transgenic mice reconstitute during B cell differentiation and subsequently undergo class switching and somatic mutation. Thus, the use of such techniques allows the production of therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar, Int. Rev. Immunol. 13: 65-93 (1995).
For a detailed discussion of this technique for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, eg, PCT Publication WO 98/24893; WO 92/01047; WO 96/34096; WO 96 / EP 0 598 877; U.S. Pat. No. 5,413,923; 5,625,126; 5,633,425; 5,569,825; No. 661,016; No. 5,545,806; No. 5,814,318; No. 5,885,793; No. 5,916,771; No. 5,939,598; See 6,075,181; and 6,114,598, which are incorporated herein by reference in their entirety. Further, Abgenix, Inc. Companies such as (Freemont, CA) and Genpharm (San Jose, CA) may be engaged in providing human antibodies against selected antigens using techniques similar to those described above.

  Fully human antibodies that recognize selected epitopes can be produced using a technique referred to as “guided selection”. In this approach, selected non-human monoclonal antibodies (eg, murine antibodies) are used to guide the selection of fully human antibodies that recognize the same epitope. (Jespers et al., Bio / technology 12: 899-903 (1988)).

(Polynucleotide encoding antibody)
The invention further provides a polynucleotide comprising a nucleotide sequence encoding the antibody and fragments thereof. The invention also relates to antibodies (preferably as defined above) that bind specifically to therapeutic proteins under stringent or lower stringency hybridization conditions, and more preferably, to the table. And a polynucleotide that hybridizes to a polynucleotide that encodes a polypeptide having the amino acid sequence of “therapeutic protein X” disclosed in the column of “SEQ ID NO: Z” of 2.

  The polynucleotide can be obtained by any method known in the art and the nucleotide sequence of the polynucleotide is determined. For example, if the nucleotide sequence of the antibody is known, the polynucleotide encoding the antibody can be constructed from chemically synthesized oligonucleotides (see, eg, Kutmeier et al., BioTechniques 17: 242 (1994)). This includes, in brief, the following steps: synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligation of these oligonucleotides, and then ligation Amplification of the resulting oligonucleotide by PCR.

Alternatively, a polynucleotide encoding an antibody can be generated from nucleic acid from a suitable source. A clone containing a nucleic acid encoding a particular antibody is not available, but if the sequence of the antibody molecule is known, the nucleic acid encoding the immunoglobulin can be chemically synthesized or can be synthesized by a suitable source ( For example, an antibody cDNA library, or a cDNA library generated from any tissue or cell that expresses the antibody (eg, a hybridoma cell selected for expression of the antibody), or a nucleic acid isolated therefrom (preferably PolyA + RNA)), for example, by PCR amplification using synthetic primers hybridizable to the 3 ′ and 5 ′ ends of the sequence to identify cDNA clones from the cDNA library encoding the antibody, or Cloning using oligonucleotide probes specific for specific gene sequences It may be obtained by ring. The amplified nucleic acid generated by PCR can then be cloned into a replicable cloning vector using any method well known in the art (see Example 107).

  Once the nucleotide sequence of an antibody and the corresponding amino acid sequence are determined, the nucleotide sequence of the antibody can be determined using methods well known in the art for manipulation of nucleotide sequences (eg, recombinant DNA techniques, site-directed mutagenesis, PCR, etc. (See, for example, Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY and Ausubel et al. Which are both incorporated herein by reference in their entirety))). There are operated, for example, to produce an antibody which has an amino acid sequence which differs as to produce amino acid substitutions, deletions, and / or insertions.

  In certain embodiments, the amino acid sequences of the heavy and / or light chain variable domains are determined by methods well known in the art (eg, sequence hypervariability) for identification of complementarity determining region (CDR) sequences. Other heavy and light chain variable regions can be examined by comparing them to known amino acid sequences). Using conventional recombinant DNA technology, one or more CDRs can be inserted into the framework region as described above (eg, into the human framework region to humanize non-human antibodies). . This framework region can be naturally occurring or can be a consensus framework region, and preferably can be a human framework region (see, eg, Chothia et al., J. Mol. Biol.278: 457-479 (1998)). Preferably, the polynucleotide produced by the combination of framework regions and CDRs encodes an antibody that specifically binds to a polypeptide of the invention. Preferably, as discussed above, one or more amino acid substitutions can be made within the framework regions, and preferably the amino acid substitution improves the binding of the antibody to its antigen. In addition, such methods also provide for amino acid substitutions or deletions of one or more variable region cysteine residues involved in intrachain disulfide bonds to produce antibody molecules that lack one or more intrachain disulfide bonds. Can be used to make. Other changes to the polynucleotide are encompassed by the present invention and within the skill of the art.

  Furthermore, a technique developed to produce “chimeric antibodies” by splicing genes from appropriate antigen-specific mouse antibody molecules with genes from appropriate biologically active human antibody molecules (Morrison) Et al., Proc. Natl. Acad. Sci. 81: 851-855 (1984); As described above, a chimeric antibody is a molecule in which different portions are derived from different animal species, and such molecules have variable regions derived from mouse mAb and human immunoglobulin constant regions (eg, humanized antibodies). .

  Alternatively, the described techniques for the production of single chain antibodies (US Pat. No. 4,946,778; Bird, Science 242: 423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879. -5883 (1988); and Ward et al., Nature 334: 544-54 (1989)) can be adapted for the production of single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. E. Techniques for the assembly of functional Fv fragments in E. coli can also be used (Skerra et al., Science 242: 1038-1041 (1988)).

(Recombinant expression of antibody)
Recombinant expression of an antibody, or a fragment, derivative or analog thereof (eg, an antibody heavy or light chain or single chain antibody) requires the construction of an expression vector containing a polynucleotide encoding the antibody. Once a polynucleotide of the invention encoding an antibody molecule or heavy or light chain of an antibody, or a portion thereof (preferably containing a heavy or light chain variable domain) is obtained, production of the antibody molecule Vectors for can be generated by recombinant DNA techniques using techniques well known in the art. Accordingly, methods for preparing a protein by expression of a polynucleotide containing a nucleotide sequence encoding an antibody are described herein. Methods well known to those skilled in the art can be used for the construction of expression vectors containing sequences encoding the antibody and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Accordingly, the present invention relates to a replicable vector comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a variable domain of a heavy or light chain, operably linked to a promoter. I will provide a. Such vectors can comprise a nucleotide sequence encoding the constant region of the antibody molecule (see, eg, PCT publication WO86 / 05807; PCT publication WO89 / 01036; and US Pat. No. 5,122,464), The variable domain of the antibody can then be cloned into such a vector for the entire expression of the heavy or light chain.

  The expression vector is transferred into a host cell by conventional techniques, and the transfected cells are then cultured by conventional techniques to produce antibodies. Accordingly, the present invention includes a host cell comprising a polynucleotide encoding an antibody of the present invention, or a heavy or light chain thereof, or a single chain antibody, operably linked to a heterologous promoter. In a preferred embodiment for the expression of a double chain antibody, vectors encoding both heavy and light chains are co-expressed in a host cell for expression of the entire immunoglobulin molecule, as detailed below. Can be done.

  A variety of host expression vector systems may be utilized to express the antibody molecules of the invention. Such a host expression system represents a vehicle in which the coding sequence of interest can be produced and subsequently purified, but is also present in situ when transformed or transfected with a sequence encoding the appropriate nucleotide. Represents a cell capable of expressing an antibody molecule of the invention. These include, but are not limited to: bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody-encoding sequences (eg, E. coli, A microorganism such as B. subtilis; a yeast transformed with a recombinant yeast expression vector comprising a sequence encoding an antibody (eg, Saccharomyces, Pichia); a recombinant viral expression vector comprising a sequence encoding an antibody (eg, Insect cell lines infected with baculoviruses; Plant cell lines infected with recombinant viral expression vectors (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or recombinant plasmid expression vectors containing sequences encoding antibodies ( example Plant cell lines transformed with the Ti plasmid); or a promoter derived from the genome of a mammalian cell (eg, a metallothionein promoter) or a promoter derived from a mammalian virus (eg, an adenovirus late promoter; vaccinia virus); A mammalian cell line carrying a recombinant expression construct comprising a 5K promoter) (eg COS, CHO, BHK, 293, 3T3 cells). Preferably, bacterial cells such as Escherichia coli, and more preferably eukaryotic cells are used for the expression of recombinant antibody molecules, particularly for the expression of whole recombinant antibody molecules. For example, mammalian cells such as Chinese hamster ovary cells (CHO) in combination with vectors such as the major immediate early gene promoter element from human cytomegalovirus are effective for antibodies. Expression system (Foecking et al., Gene 45: 101 (1986); Cockett et al., Bio / Technology 8: 2 (1990)).

  In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, if large quantities of such proteins are to be produced, vectors directed to high level expression of easily purified fusion protein products may be desired for the production of pharmaceutical compositions of antibody molecules. Such vectors include, but are not limited to, the following: the antibody-encoding sequence can be individually linked in-frame to the vector along with the lacZ-encoding region, resulting in a fusion protein. Is produced. E. coli expression vector pUR278 (Ruther et al., EMBO J. 2: 1791 (1983)); pIN vector (Inouye & Inouye, Nucleic Acids Res. 13: 3101-3109 (1985); 5509 (1989)). pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can be readily purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vector is designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

  In insect systems, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express heterologous genes. This virus grows in Spodoptera frugiperda cells. The sequence encoding the antibody can be individually cloned into a non-essential region of the virus (eg, the polyhedrin gene) and placed under the control of an AcNPV promoter (eg, the polyhedrin promoter).

  In mammalian host cells, a number of viral-based expression systems can be utilized. In cases where an adenovirus is used as an expression vector, the sequence encoding the antibody of interest can be ligated to an adenovirus transcription / translation control complex, eg, the late promoter and three-part leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region (eg, E1 or E3 region) of the viral genome results in a recombinant virus that is viable and capable of expressing antibody molecules in the infected host (eg, Logan & Shenk, Proc. Natl. Acad). Sci.USA 81: 355-359 (1984)). A specific initiation signal may also be required for efficient translation of the sequence encoding the inserted antibody. These signals include the ATG start codon and adjacent sequences. In addition, this initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., Methods in Enzymol. 153: 51-544 (1987)).

  In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (eg, glycosylation) and processing (eg, cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. For this purpose, eukaryotic host cells with cellular mechanisms for the precise processing of the first transcription, glycosylation and phosphorylation of the gene product can be used. Such mammalian host cells include CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, WI38, and particularly breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and For example, but not limited to normal breast cell lines such as CRL7030 and Hs578Bst.

  Long-term high-yield production and stable expression of recombinant proteins are preferred. For example, cell lines that stably express antibody molecules can be engineered. Rather than using an expression vector containing a viral origin of replication, the host cell may be controlled by appropriate expression control elements (eg, promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.), and selectable markers. Can be transformed. Following the introduction of the heterologous DNA, the engineered cells can be grown in enriched medium for 1-2 days and then switched to a selective medium. The selectable marker in the recombinant plasmid confers resistance to selection and the cell stably integrates the plasmid into its chromosome and proliferates to form a cell growth foci that can then be cloned and proliferated. To become a cell line. This method can be advantageously used to engineer cell lines that express antibody molecules. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.

  A number of selection systems can be used, including herpes simplex virus thymidine kinase (Wigler et al., Cell 11: 223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48: 202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22: 817 (1980)) genes (these genes can be used in tk-, hgprt- or aprt-cells, respectively). Including but not limited. Antimetabolite resistance can also be used as a basis for selection of the following genes: dhfr, which confer resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77: 357 (1980); O'Hare Proc. Natl. Acad. Sci. USA 78: 1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78: 2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 (Clinical Pharmacy 12: 488-505; Wu and Wu, Biotherapy 3: 87-95 (1991); Tolstoshev, Ann. Re. Pharmacol.Toxicol.32: 573-596 (1993); Mulligan, Science 260: 926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem.62: 191-217 (1993); , TIB TECH 11 (5): 155-215); as well as hygro, which confers resistance to hygromycin (Santerre et al., Gene 30: 147 (1984)). Methods generally known in the field of recombinant DNA technology can be routinely applied to select the desired recombinant clone, and such methods are described below: For example, Ausubel et al. ( Ed.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Express, H.c.Nr. Chapters and Chapters 13, John Wiley & Sons, NY (1994); in et al., J. Mol. Biol. 150: 1 (1981), which are incorporated herein by reference in their entirety.

  The level of expression of antibody molecules can be increased by vector amplification (reviewed by Bebbington and Hentschel, The use of vectors based on gene expression for the clone of genes in Ammelin. New York, 1987)). If the marker in the vector system expressing the antibody is amplifiable, an increase in the level of inhibitor present in the host cell culture will increase the number of copies of the marker gene. Since the amplified region is linked to the antibody gene, antibody production is also increased (Crouse et al., Mol. Cell. Biol. 3: 257 (1983)).

  Vectors that use glutamine synthase (GS) or DHFR as selectable markers can be amplified in the presence of the drugs methionine sulphoximine or methotrexate, respectively. An advantage of a glutamine synthase-based vector is the availability of a glutamine synthase negative cell line (eg, NS0 of the mouse myeloma cell line). The glutamine synthase expression system can also function in glutamine synthase expressing cells, such as Chinese hamster ovary (CHO) cells, by providing additional inhibitors to prevent endogenous gene function. The glutamine synthase expression system and its components are described in detail in the following PCT publications: WO 87/04462; WO 86/05807; WO 89/01036; WO 89/10404 and WO 91/06657, which are herein incorporated by reference in their entirety. Incorporated by reference). Furthermore, glutamine synthase expression vectors that can be used in accordance with the present invention are described, for example, in Lonza Biologics, Inc. Commercially available from vendors including (Portsmouth, NH). Monoclonal antibody expression and production using the GC expression system in mouse myeloma cells is described in Bebbington et al., Bio / technology 10: 169 (1992) and Biblia and Robinson, Biotechnol. Prog. 11: 1 (1995), which are hereby incorporated by reference in their entirety.

  A host cell can be co-transfected with two expression vectors of the invention, a first vector encoding a heavy chain derived polypeptide and a second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers that allow equal expression of heavy and light chain polypeptides. Alternatively, a single vector can be used that encodes and can express both heavy and light chain polypeptides. In such situations, the light chain should be placed in front of the heavy chain to avoid excessive toxic free heavy chains (Proudfoot, Nature 322: 52 (1986); Kohler, Proc. Natl. Acad. Sci.USA 77: 2197 (1980)). Coding sequences for heavy and light chains can include cDNA or genomic DNA.

  Once the antibody molecule of the invention is produced by an animal, chemically synthesized, or recombinantly expressed, any method for purification of immunoglobulin molecules known in the art, For example, chromatography (eg, ion exchange, affinity (especially with protein A followed by affinity for specific antigen), and size column chromatography), centrifugation, differential solubility, or any other standard for protein purification Can be purified by conventional techniques. Further, an antibody or fragment thereof that binds to a therapeutic protein and can respond to the therapeutic protein portion of an albumin fusion protein of the invention is described herein or otherwise known in the art, It can be fused to a heterologous polypeptide sequence to facilitate purification.

(Modification of antibody)
Antibodies, or fragments or variants, that bind to the therapeutic protein can be fused to a marker sequence, such as a peptide that facilitates purification. In a preferred embodiment, the marker amino acid sequence is inter alia a hexa-histidine peptide (eg the tag provided in the pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91111), and many of these markers. The amino acid sequence is commercially available. For example, Gentz et al., Proc. Natl. Acad. Sci. USA 86: 821-824 (1989), hexa-histidine provides convenient purification of the fusion protein. Other peptide tags useful for purification are hemagglutinin tags (also referred to as “HA” tags) corresponding to epitopes from influenza hemagglutinin protein (Wilson et al., Cell 37: 767 (1984)). , And “flag” tags, but not limited to.

  The invention further includes an antibody or fragment thereof conjugated to a diagnostic or therapeutic agent. The antibodies can be used diagnostically to monitor tumor development or progression, for example, as part of a clinical testing procedure (eg, to determine the efficacy of a given treatment regimen). Detection can be facilitated by linking the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomography, and non-radioactive paramagnetic metal ions Is mentioned. This detectable substance can be either directly to the antibody (or fragment thereof), or indirectly via a mediator using techniques known in the art (eg, linkers known in the art, etc.). It can be linked or conjugated. See, for example, US Pat. No. 4,741,900 for metal ions that can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic complex include streptavidin / biotin and avidin / biotin; Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; examples of luminescent materials include luminol; bioluminescent materials Examples of include luciferase, luciferin and aequorin; and examples of suitable radioactive materials include 125I, 131I, 111In or 99Tc. Other examples of detectable substances are described elsewhere in this specification.

  Further, the antibodies of the present invention may be used in therapeutic moieties (eg, cytotoxins (eg, cytostatic or cell killing agents)), therapeutic agents or radiometal ions (eg, α-emitters (eg, 213Bi)). Can be combined. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, dianthorubicin d anthracin dione), mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs thereof. Therapeutic agents include antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine, alkylating agents (eg, mechlorethamine, thioepa chlorambucil, mel Faran, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum (II) (DDP) cisplatin, anthracyclines (eg, Daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (eg dactinomycin ( Before actinomycin are), bleomycin, mithramycin, and anthramycin, (Anthramycin) (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine), including, but not limited to.

  The conjugates of the invention can be used to modify a given biological response, and the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety can be a protein or polypeptide having the desired biological activity. Examples of such proteins include toxins (eg, abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin); proteins (eg, tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet-derived growth factor) Tissue plasminogen activator, apoptotic drugs (eg, TNF-α, TNF-β, AIM I (see WO 97/33899), AIM II (see WO 97/34911) , Fas ligand (Takahashi et al., Int. Immunol. 6: 1567-1574 (1994)), VEGI (see WO 99/23105)), thrombotic drugs or antiangiogenic drugs (eg Angio) Statin or endostatin) Or biological response modifiers (eg lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophages Colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors).

  The antibody can also be attached to a solid support, which is particularly useful for immunoassay or purification of the target antigen. Such solid supports include but are not limited to glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

  Techniques for coupling such therapeutic moieties to antibodies are well known, see, eg, Arnon et al., “Monoclonal Antibodies For Immunology Of Drugs In Cancer Therapies, Monoclonal Antibodies et al. R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery” Controlled Drug Delivery (2nd edition), Robinson et al. (Ed.), Pp. 623-53 (Marcel Deker, Inc., Ah. Carriers Of Cyto oxic Agents In Cancer Therapy: A Review "Monoclonal Antibodies'84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985);" Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapeutics "Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (Ed.), Pages 303-16 (Academic Press 1985), and Thorpe et al. "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev. 62: 119-58 (1982).

  Alternatively, the antibody is conjugated to a secondary antibody and the heterologous conjugate of antibody (as described by Segal in US Pat. No. 4,676,980, which is incorporated by reference herein in its entirety) ( heteroconjugate).

  An antibody with or without a therapeutic moiety that binds to the antibody administered alone or in combination with cytotoxic factors and / or cytokines can be used as a therapeutic agent.

(Antibody-albumin fusion)
The antibody that binds to the therapeutic protein and can correspond to the therapeutic protein portion of the albumin fusion protein of the present invention includes an antibody that binds to the therapeutic protein disclosed in the column of “Therapeutic protein X” in Table 1, or a fragment or Although a modification is mentioned, it is not limited to these.

  In certain embodiments, an antibody fragment or variant that immunospecifically binds to a therapeutic protein and corresponds to a therapeutic protein portion of an albumin fusion protein comprises or consists of a VH domain. In other embodiments, the fragment or variant of an antibody that immunospecifically binds to a therapeutic protein and corresponds to the therapeutic protein portion of an albumin fusion protein comprises one, two, or three VH CDRs, or these Consists of. In other embodiments, the fragment or variant of an antibody that immunospecifically binds to a therapeutic protein and corresponds to the therapeutic protein portion of an albumin fusion protein comprises or consists of VH CDR1. In other embodiments, an antibody fragment or variant that immunospecifically binds to a therapeutic protein and corresponds to a therapeutic protein portion of an albumin fusion protein comprises or consists of VH CDR2. In other embodiments, an antibody fragment or variant that immunospecifically binds to a therapeutic protein and that corresponds to a therapeutic protein portion of an albumin fusion protein comprises or consists of VH CDR3.

  In certain embodiments, an antibody fragment or variant that binds immunospecifically to a therapeutic protein and corresponds to a therapeutic protein portion of an albumin fusion protein comprises or consists of a VL domain. In other embodiments, an antibody fragment or variant that immunospecifically binds to a therapeutic protein and corresponds to a therapeutic protein portion of an albumin fusion protein comprises one, two, or three VL CDRs, or these Consists of. In other embodiments, an antibody fragment or variant that immunospecifically binds to a therapeutic protein and corresponds to a therapeutic protein portion of an albumin fusion protein comprises or consists of VL CDR1. In other embodiments, an antibody fragment or variant that immunospecifically binds to a therapeutic protein and corresponds to a therapeutic protein portion of an albumin fusion protein comprises or consists of VL CDR2. In other embodiments, the fragment or variant of an antibody that immunospecifically binds to a therapeutic protein and corresponds to the therapeutic protein portion of an albumin fusion protein comprises or consists of VL CDR3.

  In other embodiments, the antibody fragment or variant that immunospecifically binds to a therapeutic protein and corresponds to the therapeutic protein portion of an albumin fusion protein is one, two, three, four, five or Contains or consists of 6 VL CDRs and / or VH CDRs.

In a preferred embodiment, a fragment or variant of an antibody that immunospecifically binds to a therapeutic protein and corresponds to a therapeutic protein portion of an albumin fusion protein is a peptide linker (eg, (Gly ₄ Ser) ₃ (SEQ ID NO: 1092) The scFv containing or consisting of the VH domain of the therapeutic antibody linked to the VL domain of the therapeutic antibody by

(Immunophenotyping)
An albumin fusion protein of the invention comprising at least one fragment or variant of an antibody that binds to an antibody of the invention, or a therapeutic protein (or fragment or variant thereof) is an immunophenotyping of cell lines and biological samples. Can be used for. The therapeutic proteins of the present invention may be useful as cell-specific markers, or more specifically as cell markers that are differentially expressed at various stages of differentiation and / or maturation of a particular cell type. A monoclonal antibody directed against a specific epitope, or combination of epitopes (or a rubmin fusion protein comprising at least one fragment or variant of an antibody that binds to a therapeutic protein) can be used to screen a population of cells that express the marker. Make it possible. Various techniques can be utilized with monoclonal antibodies (or rubmin fusion proteins comprising at least one fragment or variant of an antibody that binds to a therapeutic protein) to screen a cell population that expresses the marker, and The techniques include magnetic separation using magnetic beads coated with antibodies, “panning” using antibodies attached to a solid matrix (ie, plate), and flow cytometry (eg, US Pat. No. 5,985,660). And Morrison et al., Cell, 96: 737-49 (1999)).

  These techniques are “non-self” in transplantation to prevent hematological malignancies (ie, minimal residual disease (MRD) in acute leukemia patients) and graft-versus-host disease (GVHD). “Allows screening of specific populations of cells as can be found with the cells. Alternatively, these techniques allow for the screening of hematopoietic stem and progenitor cells that can undergo proliferation and / or differentiation as can be found in human umbilical cord blood.

Characterization of albumin fusion proteins comprising antibodies that bind to therapeutic proteins and fragments or variants of antibodies that bind to therapeutic proteins
An albumin fusion protein of the invention comprising at least a fragment or variant of an antibody that binds to an antibody of the invention, or a therapeutic protein (or fragment or variant thereof) can be characterized in a variety of ways. In particular, an albumin fusion protein of the invention comprising at least a fragment or variant of an antibody that binds to a therapeutic protein can be obtained using techniques described herein or routinely known in the art. Can be assayed for the ability to specifically bind to the same antigen that is specifically bound by the antibody that binds to the therapeutic protein, corresponding to the antibody that binds to the therapeutic protein portion of the albumin fusion protein.

  The ability of an albumin fusion protein of the invention comprising at least a fragment or variant of an antibody that binds to an antibody of the invention, or a therapeutic protein (or fragment or variant thereof) to (specifically) bind to a specific protein or epitope. Assays for can be performed in solution (eg, Houghten, Bio / Techniques 13: 412-421 (1992)) or beads (eg, Lam, Nature 354: 82-84 (1991)) on a chip (eg, Fodor, Nature 364: 555-556 (1993)) on bacteria (eg, US Pat. No. 5,223,409) and spores (eg, US Pat. No. 5,571,698; 484 and 5,223,409) Plasmids (eg, Cull et al., Proc. Natl. Acad. Sci. USA 89: 1865-1869 (1992)) or phages (eg, Scott and Smith, Science 249: 386-390 (1990); Devlin, Science 249: 404 Cirla et al., Proc. Natl. Acad. Sci. USA 87: 6378-6382 (1990); Felici, J. Mol. Biol. 222: 301-310 (1991)) (of these references). Each may be implemented in its entirety herein incorporated by reference. An albumin fusion protein of the invention comprising an antibody of the invention, or at least a fragment or variant of an antibody that binds to a therapeutic protein (or fragment or variant thereof) is also a technique described herein or other such They can be assayed for their specificity and affinity for specific proteins or epitopes using techniques known in the art or routinely modified.

  An albumin fusion protein of the present invention comprising at least a fragment or variant of an antibody that binds to a therapeutic protein may correspond to other antigens (eg, the therapeutic protein portion of the albumin fusion protein of the present invention) by any method known in the art. Can be assayed for cross-reactivity with molecules that are specifically bound by antibodies that bind to therapeutic proteins (or fragments or variants thereof) with molecules that have sequence conservation / structure conservation.

  Immunoassays that can be used to analyze (immunospecific) binding and cross-reactivity include, but are not limited to, Western blots, radioimmunoassays, ELISAs (enzyme-linked immunosorbent assays), Competition using techniques such as “sandwich” immunoassay, immunoprecipitation assay, precipitation reaction, gel diffusion precipitation reaction, immunodiffusion assay, aggregation assay, complement binding assay, immunoradiometric assay, fluorescent immunoassay, protein A immunoassay Examples include but are not limited to assay systems and non-competitive assay systems. Such assays are conventional and well known in the art (see, eg, Ausubel et al., 1994, Current Protocols in Molecular Biology, Vol. 1, which is incorporated herein by reference in its entirety. See John Wiley & Sons, Inc., New York). Exemplary immunoassays are briefly described below (although these are not intended to be limiting).

Immunoprecipitation protocols generally involve RIPA buffer (1% NP-40 or Triton X-100, 1% deoxychol) supplemented with protein phosphatase inhibitors and / or protease inhibitors (eg, EDTA, PMSF, aprotinin, sodium vanadate). Lysing a population of cells in a lysis buffer such as sodium acid, 0.1% SDS, 0.15M NaCl, 0.01M sodium phosphate (pH 7.2), 1% Trasylol), Adding an albumin fusion protein of the present invention comprising at least one fragment or variant of an antibody that binds to an antibody or therapeutic protein (or fragment or variant thereof) to a cell lysate, for a period of time (eg, 1 to 4 hours) ) Ink at 40 ° C Activating, protein A sepharose beads and / or protein G sepharose beads (or, if the albumin fusion protein comprises at least one fragment or variant of a therapeutic antibody, coated with an appropriate anti-idiotype antibody or anti-albumin antibody Adding the beads) to the cell lysate, incubating at 40 ° C. for about 1 hour or more, washing the beads in lysis buffer, and resuspending the beads in SDS / sample buffer. To do.
The ability of the antibodies or albumin fusion proteins of the present invention to immunoprecipitate a particular antigen can be assayed, for example, by Western blot analysis. Those skilled in the art will appreciate that parameters that can be modified to increase the binding of the antibody or albumin fusion protein to the antigen and to reduce the background (eg, pre-cleaning the cell lysate using Sepharose beads). There may be knowledge about. For further discussion on immunoprecipitation protocols, see, eg, Ausubel et al., 1994, Current Protocols in Molecular Biology, Volume 1, John Wiley & Sons, Inc. , New York, 10.16.1.

Western blot analysis generally involves preparing a protein sample, electrophoresis of the protein sample in a polyacrylamide gel (eg, 8% -20% SDS-PAGE depending on the molecular weight of the antigen), Transferring from a polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in a blocking solution (eg, PBS with 3% BSA or nonfat dry milk), washing buffer (eg, PBS- Washing the membrane in Tween 20), applying the antibody or albumin fusion protein of the invention (diluted in blocking buffer) to the membrane, washing the membrane in washing buffer, blocking buffer Enzyme substrate diluted in liquid (eg horseradish Peroxidase or alkaline phosphatase) or radioactive molecule (e.g., ³² to recognize the ^P or ^{125 I)} secondary antibody conjugated to a (primary antibody, for example, applying an anti-human serum albumin antibody), its in washing buffer Washing the membrane as well as detecting the presence of the antigen. One skilled in the art may be aware of parameters that can be modified to increase the detected signal and to reduce background noise. For further discussion on Western blot protocols, see, eg, Ausubel et al., 1994, Current Protocols in Molecular Biology, Volume 1, John Wiley & Sons, Inc. , New York, 10.8.1.

  ELISA comprises the steps of preparing an antigen, coating a well of a 96-well microtiter plate with the antigen, washing away the antigen that did not bind to the well, enzyme substrate (eg, horseradish peroxidase or alkaline phosphatase) Adding an antibody of the invention or albumin fusion protein (including at least one fragment or variant of an antibody that binds to a therapeutic protein) conjugated to a detectable compound such as to the well and incubating for a period of time; Including washing away unbound albumin fusion protein or non-specifically bound albumin fusion protein and detecting the presence of albumin fusion protein that specifically binds to the antibody or well coating antigen. That. In an ELISA, the antibody or albumin fusion protein need not be bound to a detectable compound; instead, a second antibody (recognizing the antibody or albumin fusion protein, respectively) bound to the detectable compound Can be added to the wells. Further, instead of coating the well with the antigen, the antibody or albumin fusion protein can be coated to the well. In this case, the detectable molecule can be an antigen conjugated to a detectable compound, such as an enzyme substrate (eg, horseradish peroxidase or alkaline phosphatase). One skilled in the art may be aware of parameters that can be modified to increase the detected signal, and other variations of ELISA known in the art. For further discussion on ELISA, see, eg, Ausubel et al., 1994, Current Protocols in Molecular Biology, Volume 1, John Wiley & Sons, Inc. , New York, 11.2.1.

The binding affinity of the albumin fusion protein for the protein, antigen or epitope and the off-rate of the antibody- or albumin fusion protein-protein / antigen / epitope interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay, which is an antibody or albumin fusion of the present invention in the presence of a labeled antigen (eg, ³ H or ¹²⁵ I) and increasing amounts of unlabeled antigen. Incubation with protein and detection of antibody bound to labeled antigen. The affinity of an antibody or albumin fusion protein of the invention for a particular protein, antigen or epitope, and binding off-rate can be determined from data by Scatchard plot analysis. Competition of the antibody or albumin fusion protein with a second protein that binds to the same protein, antigen or epitope can also be determined using a radioimmunoassay. In this case, the protein, antigen or epitope is labeled in the presence of increasing amounts of an unlabeled second protein that binds to the same protein, antigen or epitope as the albumin fusion protein of the invention (eg, ³ H or Incubated with an antibody or albumin fusion protein of the invention conjugated to ¹²⁵ I).

  In a preferred embodiment, BIAcore kinetic analysis is used to determine the on and off rates of binding of an antibody of the invention or albumin fusion protein to a protein, antigen or epitope. BIAcore kinetic analysis can be performed using an antibody, albumin fusion protein, or specific polypeptide, antigen or antigen from a chip with a specific polypeptide, antigen or epitope, antibody or albumin fusion protein, respectively, immobilized on its surface. Analyzing the binding and dissociation of the epitope.

(Therapeutic use)
The present invention further relates to antibody-based therapies, which treat animals, preferably mammals, and most preferably human patients, to treat one or more disclosed diseases, disorders, or conditions. Administering an albumin fusion protein of the invention comprising an antibody of the invention, or at least a fragment or variant of an antibody that binds to a therapeutic protein. The therapeutic compounds of the present invention include the antibodies of the present invention (including fragments, analogs and derivatives thereof as described herein), nucleic acids encoding the antibodies of the present invention (described herein). And their fragments, analogs and derivatives and anti-idiotype antibodies), albumin fusion proteins of the invention comprising at least one fragment or variant of an antibody that binds to a therapeutic protein, and such albumin fusions Examples include, but are not limited to, nucleic acids that encode proteins. An albumin fusion protein of the invention comprising an antibody of the invention, or at least a fragment or variant of an antibody that binds to a therapeutic protein, comprises a disease, disorder or condition associated with abnormal expression and / or activity of the therapeutic protein (herein Can be used to treat, inhibit or prevent any one or more of the diseases, disorders, or conditions described therein). Treatment and / or prevention of a disease, disorder or condition associated with abnormal expression and / or activity of a therapeutic protein includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions. An albumin fusion protein of the invention comprising an antibody of the invention, or at least a fragment or variant of an antibody that binds to a therapeutic protein, is known in the art or as described herein. Can be provided in an acceptable composition.

  In specific and preferred embodiments, the present invention relates to antibody-based therapies, which include one or more diseases, disorders, or conditions, including but not limited to: neurological disorders, immune system disorders , Myopathy, reproductive disorders, gastrointestinal disorders, lung disorders, cardiovascular disorders, renal disorders, proliferative disorders and / or cancerous diseases and conditions, and / or those described elsewhere herein) To treat, an animal, preferably a mammal, and most preferably a human patient is administered an antibody of the invention, or an albumin fusion protein of the invention comprising at least a fragment or variant of an antibody that binds to a therapeutic protein. Process. Examples of the therapeutic compound of the present invention include the antibody of the present invention (for example, an antibody against a full-length protein expressed on the cell surface of a mammalian cell; an antibody against an epitope of a therapeutic protein) and a nucleic acid encoding the antibody of the present invention (here Including, but not limited to, fragments, analogs and derivatives thereof as well as anti-idiotype antibodies). An albumin fusion protein of the invention comprising an antibody of the invention, or at least a fragment or variant of an antibody that binds to a therapeutic protein, comprises a disease, disorder or condition associated with abnormal expression and / or activity of the therapeutic protein (herein Can be used to treat, inhibit or prevent any one or more of the diseases, disorders, or conditions described therein). Treatment and / or prevention of a disease, disorder or condition associated with abnormal expression and / or activity of a therapeutic protein includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions. The antibodies of the present invention are known in the art or can be provided in pharmaceutically acceptable compositions as described herein.

  An overview of how an antibody of the invention or an albumin fusion protein of the invention comprising at least a fragment or variant of an antibody that binds to a therapeutic protein can be used therapeutically includes binding the therapeutic protein locally or systemically in the body. Or binding by direct cytotoxicity of the antibody (eg, complement mediated (CDC) or effector cell mediated (ADCC)). Some of these approaches are described in more detail below. Given the teaching provided herein, one of ordinary skill in the art could, without undue experimentation, perform an antibody of the invention or albumin fusion protein of the invention comprising at least a fragment or variant of an antibody that binds to a therapeutic protein. Know how to use for diagnostic, monitoring or therapeutic purposes.

  An antibody of the invention or albumin fusion protein of the invention comprising at least a fragment or variant of an antibody that binds to a therapeutic protein may be combined with other monoclonal or chimeric antibodies or, for example, IL-2, IL-3 and IL It can be advantageously used in combination with a lymphokine such as -7 or a hematopoietic growth factor (eg, it acts to increase the number or activity of effector cells that interact with the antibody).

  An antibody of the invention or albumin fusion protein of the invention comprising at least a fragment or variant of an antibody that binds to a therapeutic protein can be used alone or in other types of treatment (eg, radiation therapy, chemotherapy, hormonal therapy, immunization, Therapeutic and anti-tumor agents). In general, administration (in the case of antibodies) of the product of a native or reactive species that is the same species as that of the patient is preferred. Accordingly, in a preferred embodiment, the human antibody, fragment derivative, analog or nucleic acid is administered to a human patient for treatment or prevention.

The use of high affinity and / or potent in vivo inhibitory and / or neutralizing antibodies against a therapeutic protein, fragment or region thereof is directly linked to the polynucleotide or polypeptide of the invention (including fragments thereof). Preferred for both immunoassay and treatment of disorders associated therewith. Such an antibody, fragment or region preferably has affinity for a polynucleotide or polypeptide of the invention (including fragments thereof). Preferred binding affinities are dissociation constants or Kd (dissociation of less than 5 × 10 ⁻² M, 10 ⁻² M, 5 × 10 ⁻³ M, 10 ⁻³ M, 5 × 10 ⁻⁴ M, 10 ⁻⁴ M Constant). Further preferred binding affinities are the same dissociation constants, or 5 × 10 ⁻⁵ M, 10 ⁻⁵ M, 5 × 10 ⁻⁶ M, 10 ⁻⁶ M, 5 × 10 ⁻⁷ M, 10 ⁻⁷ M, It contains a Kd (dissociation constant) of less than 5 × 10 ⁻⁸ M or 10 ⁻⁸ M. Further preferred binding affinities are the same dissociation constants as those, or 5 × 10 ⁻⁹ M, 10 ⁻⁹ M, 5 × 10 ⁻¹⁰ M, 10 ⁻¹⁰ M, 5 × 10 ⁻¹¹ M, 10 ^−11. M, 5 × 10 ⁻¹² M, 10 ⁻¹² M, 5 × 10 ⁻¹³ M, 10 ⁻¹³ M, 5 × 10 ⁻¹⁴ M, 10 ⁻¹⁴ M, 5 × 10 ⁻¹⁵ M, or 10 ⁻¹⁵ M Less than Kd (dissociation constant).

(Gene therapy)
In certain embodiments, a nucleic acid comprising a sequence encoding an antibody that binds to a therapeutic protein or an albumin fusion protein comprising at least a fragment or variant of an antibody that binds to the therapeutic protein is a therapeutic protein aberrant by gene therapy methods. Administered to treat, inhibit or prevent a disease or disorder associated with expression and / or activity. Gene therapy refers to a therapy performed by administering to a subject an expressed or expressible nucleic acid. In embodiments of the invention, the nucleic acids make these encoded proteins that mediate therapeutic effects.

  Any method for gene therapy available in the art can be used in accordance with the present invention. Exemplary methods are described in more detail elsewhere herein.

(Description of therapeutic or prophylactic activity)
The compounds or pharmaceutical compositions of the invention are preferably tested for the desired therapeutic or prophylactic activity in vitro prior to use in humans and then in vivo. For example, in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include the effect of the compound on cell lines or patient tissue samples. The effect of the compound or composition on the cell line and / or tissue sample can be determined utilizing techniques known to those of skill in the art, including but not limited to rosette formation assays and cell lysis assays. In vitro assays that can be used to determine whether administration of a particular compound is indicated according to the present invention include in vitro cell culture assays, in which patient tissue samples are grown in culture, The compound is then exposed or otherwise administered, and the effect of such compound on the tissue sample is observed.

(Therapeutic / prophylactic administration and composition)
The present invention provides methods of treatment, inhibition and prevention by administration of an effective amount of a compound or pharmaceutical composition of the present invention to a subject. In preferred embodiments, the compound is substantially purified (eg, substantially free of substances that limit its effect or cause undesirable side effects). The subject is preferably an animal including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, and the like, preferably mammals, and most preferably humans.

  Formulations and methods of administration that can be used when the compound comprises a nucleic acid or immunoglobulin are described above; further suitable formulations and routes of administration can be selected from those described herein below.

  Various delivery systems are known and can be used to administer a compound of the invention (eg, liposomes, microparticles, microcapsules, encapsulation in recombinant cells capable of expressing the compound, receptor-mediated Endocytosis (see, eg, Wu and Wu, J. Biol. Chem. 262: 4429-4432 (1987), construction of nucleic acids as part of retroviruses or other vectors). Introduction methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compound or composition can be administered by any convenient route (eg, by infusion or bolus injection, by absorption through the epithelium or mucosal lining (eg, oral mucosa, rectal mucosa, intestinal mucosa, etc.), and others. It can be administered with a biologically active agent. Administration can be systemic or local. In addition, the pharmaceutical compounds or compositions of the invention may include any suitable route (including intraventricular and intrathecal injection; intraventricular injection, for example, intraventricularly attached to a reservoir, such as an Ommaya reservoir. It may be desirable to introduce into the central nervous system by a catheter). Pulmonary administration can also be used, for example, by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

  In a specific embodiment, it may be desirable to administer the pharmaceutical compound or composition of the invention locally to the area in need of treatment; this is not meant to be limiting, but for example during surgery By local injection, topical application (eg in combination with a post-surgical wound dressing), by injection, by catheter, by suppository, or an implant (this implant comprises a membrane or fiber, such as a sialastic membrane) A porous, non-porous, or glue-like material). Preferably, when administering a protein of the invention, including antibodies, care must be taken to use materials that do not absorb the protein.

  In another embodiment, the compound or composition can be delivered into vesicles, particularly liposomes (Langer, Science 249: 1527-1533 (1990); Treat et al., Liposomes in the Disease of Infectious Disease and Cancer, Lope -See Berestin and Fiddler (eds.), Liss, New York, pages 353-365 (1989); Lopez-Berstein, pages 317-327; broadly see the same book).

  In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used (Langer, (supra); Sefton, CRC Crit. Ref. Biomed. Eng. 14: 201 (1987); Buchwald et al., Surgery 88: 507 (1980); Saudek et al. N. Engl. J. Med. 321: 574 (1989)). In another embodiment, polymeric materials can be used (Medical Applications of Controlled Release, Langer and Wise (ed.), CRC Pres., Boca Raton, Florida (1974); Controlled DrugDrivable Drug Bioavailability. And Ball (Ed.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); Levy et al., Science 228: 190 (1985). ); During et al., Ann. 25: 351 (1989); see also Howard et al., J. Neurosurg. 71: 105 (1989)). In yet another embodiment, the controlled release system can be placed near a therapeutic target, eg, the brain, and therefore requires only a portion of the systemic dose (eg, Goodson, Medical Applications of Controlled Release, (Supra), Vol. 2, pages 115-138 (1984)).

  Other controlled release systems are discussed in a review by Langer (Science 249: 1527-1533 (1990)).

  In a specific embodiment, where the compound of the invention is a nucleic acid encoding a protein, the nucleic acid is configured as part of a suitable nucleic acid expression vector and is administered so that it is intracellular. (Eg, by use of retroviral vectors (see US Pat. No. 4,980,286)) or by direct injection or by use of microparticle bombardment (eg, gene gun; Biolistic, Dupont), or Administration by coating with lipids or cell surface receptors or transfection agents, or in combination with homeobox-like peptides known to enter the nucleus (eg, Joliot et al., Proc. Natl. Acad. Sci. USA). 88: 1864-1868 (19 (See 1)) etc., or can be administered in vivo to promote expression of the encoded protein, or the nucleic acid can be introduced into the cell and by homologous recombination for expression. It can be integrated into the host cell DNA.

The present invention also provides a pharmaceutical composition. Such compositions comprise a therapeutically effective amount of the compound, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” is recognized by federal regulatory authorities or the US government for use in animals, and more particularly in humans, or the US Pharmacopoeia or other Means listed in the generally accepted pharmacopoeia. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers include sterile liquids, such as water and oils, including oils of petroleum, animal, vegetable or synthetic origin (eg, peanut oil, soybean oil, mineral oil, sesame oil). It can be.
Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene , Glycol, water, ethanol and the like. The composition can also include minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate and the like. Examples of suitable pharmaceutical carriers are E. W. Described in “Remington's Pharmaceutical Sciences” by Martin. Such compositions comprise a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the dosage form.

  In a preferred embodiment, the composition is formulated as a pharmaceutical composition employed for intravenous administration to humans according to conventional procedures. Typically, compositions for intravenous administration are sterile isotonic aqueous buffer solutions. Where necessary, the composition may also include a solubilizer and a local anesthetic such as lignocaine that relieves pain at the site of the injection. In general, the ingredients are either separately or mixed together in a single dosage form, eg, in a sealed container such as an ampoule or sachet that represents a quantity of the active agent. Lyophilized powder or water free concentrate. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

  The compounds of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include salts formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and the like, and sodium, potassium, ammonium, calcium, ferric hydroxide, And salts formed with cations such as those derived from isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine and the like.

  The amount of a compound of the invention that is effective in this treatment (suppression and prevention of a disease or disorder associated with aberrant expression and / or activity of a polypeptide of the invention) can be determined by standard clinical techniques. In addition, in vitro assays can be used, if necessary, to help identify optimal dosage ranges. The exact dose to be used in the formulation will also depend on the route of administration, and the severity of the disease or disorder, and should be determined according to the judgment of the practitioner and the circumstances of each patient. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

  For antibodies, the dosage administered to a patient is typically 0.1 mg / kg to 100 mg / kg of the patient's body weight. Preferably, the dosage administered to the patient is between 0.1 and 20 mg / kg of the patient's body weight, more preferably 1 mg to 10 mg / kg of the patient's body weight. In general, human antibodies have a longer half-life in the human body than antibodies from other species due to immune responses to foreign polypeptides. Thus, low dosages and infrequent administration of human antibodies is often possible. Furthermore, the dosage and frequency of administration of the antibodies of the invention can be reduced by enhancing antibody uptake and tissue penetration (eg, into the brain) by modification (eg, lipidation). .

(Diagnosis and imaging)
Labeled antibodies that bind to therapeutic proteins (or fragments or variants thereof) (including albumin fusion proteins that include at least fragments or variants of antibodies that bind to therapeutic proteins), and derivatives and analogs thereof are for diagnostic purposes. Can be used to detect, diagnose or monitor diseases, disorders or / and conditions associated with abnormal expression and activity of therapeutic proteins. The present invention provides for the detection of aberrant expression of a therapeutic protein, which comprises: (a) using one or more antibodies specific for the polypeptide of interest, to detect the therapeutic protein in a cell or body fluid of an individual. Assayed polypeptide gene comprising: assaying expression; and (b) comparing the level of gene expression to the level of standard gene expression, thereby comparing to the standard expression level An increase or decrease in expression level indicates abnormal expression.

  The present invention provides a diagnostic assay for diagnosing a disorder, the assay comprising: (a) one specific for a therapeutic protein or albumin fusion protein comprising a fragment of at least an antibody variant specific for the therapeutic protein. Assaying the expression of therapeutic protein in an individual's cells or body fluids using the above antibodies, and (b) comparing the level of gene expression to the level of standard gene expression, An increase or decrease in the assayed therapeutic protein gene expression level compared to its standard expression level indicates a particular disorder. With respect to cancer, the presence of a relatively high amount of transcript in an individual's biopsy tissue may indicate a predisposition for the development of the disease or provide a means for detecting the disease before the appearance of actual clinical symptoms. Can be provided. This type of more definitive diagnosis may allow health professionals to use preventive measures, or allow early aggressive treatment, thereby preventing the development or further progression of cancer.

  Albumin fusion proteins comprising at least a fragment of an antibody of the invention or a variant of an antibody specific for a therapeutic protein are used in biological samples using classical immunohistological methods known to those skilled in the art. Protein levels can be assayed (see, for example, Jalkanen et al., J. Cell. Biol. 101: 976-985 (1985); Jalkanen et al., J. Cell. Biol. 105: 3087-3096 (1987)). . Other methods based on other antibodies useful for detecting protein gene expression include immunoassays, such as enzyme-linked immunosorbent assays (ELISA) and radioimmunoassays (RIA). Suitable antibody assay labels are known in the art and are enzyme labels (eg, glucose oxidase); radioisotopes (eg, iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H ), Indium (112In), and technetium (99Tc)); luminescent labels (eg, luminol); and fluorescent labels (eg, fluorescein and rhodamine), and biotin.

  One aspect of the invention is the detection and diagnosis of diseases or disorders associated with abnormal expression of therapeutic proteins in animals, preferably mammals, and most preferably humans. In one embodiment, diagnosis comprises a) administering to a subject (eg, parenterally, subcutaneously or intraperitoneally) an effective amount of a labeled molecule that specifically binds to the polypeptide of interest. B) to allow preferential enrichment of the labeled molecule at the site in the subject where the therapeutic protein is expressed (and to remove unbound labeled molecule to background levels); A) waiting for a time interval after administration; c) determining a background level; and d) detecting a labeled molecule in the subject so that the label above the background level is exceeded. Detection of the activated molecule indicates that the subject has a particular disease or disorder associated with abnormal expression of the therapeutic protein. The background level can be determined by a variety of methods including comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.

  It will be appreciated in the art that the size of the subject and the imaging system used will determine the amount of imaging moiety needed to produce a diagnostic image. In the case of a radioisotope moiety, for a human subject, the amount of radioactivity injected will typically range from about 5 to 20 millicuries of 99mTc. The albumin fusion protein comprising at least a fragment or variant of the antibody that binds to the labeled antibody, antibody fragment or therapeutic protein is then preferentially accumulated at the location of the cell containing the particular therapeutic protein. In vivo tumor imaging is described in S.H. W. Burchiel et al., “Immunopharmacologicals of Radiolabeled Antibodies and Their Fragments” (Tumor Imaging: The Radio Detection of Cancer. Chapter 13A. .

  Depending on a number of variables, including the type of label used and the mode of administration, the labeled molecules preferentially concentrate at the site of the subject and unbound labeled molecules are background The time interval after administration that allows it to be cleared to a level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment, the time interval after administration is 5 to 20 days or 5 to 10 days.

  In one embodiment, monitoring a disease or disorder involves repeating a method for diagnosing the disease or disorder (eg, 1 month after initial diagnosis, 6 months after initial diagnosis, 1 year after initial diagnosis). Etc.).

  The presence of the labeled molecule can be detected from the patient using methods known in the art for in vivo scanning. These methods depend on the type of label used. One skilled in the art can determine an appropriate method for detecting a particular label. Methods and devices that can be used in the diagnostic methods of the present invention include, but are not limited to, whole body scanning such as computed tomography (CT), proton emission tomography (PET), and magnetic resonance imaging (MRI). , And ultrasound diagnostic methods.

  In certain embodiments, the molecule is labeled with a radioisotope and detected from the patient using a radiation responsive surgical instrument (Thurston et al., US Pat. No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and detected from the patient using a fluorescence responsive scanning instrument. In another embodiment, the molecule is labeled with a positron emitting metal and detected from the patient using proton emission tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and detected from the patient using magnetic resonance imaging (MRI). An antibody that specifically detects albumin fusion protein but not albumin or therapeutic protein alone is a preferred embodiment. These can be used to detect albumin fusion proteins as described throughout the specification.

(kit)
The present invention provides kits that can be used in the above methods. In one embodiment, the kit comprises an antibody (preferably a purified antibody) in one or more containers. In certain embodiments, the kit of the invention comprises a substantially isolated polypeptide comprising an epitope that is specifically immunoreactive with an antibody included in the kit. Preferably, the kit of the present invention further comprises a control antibody that does not react with the target polypeptide. In another specific embodiment, the kit of the invention comprises the binding of an antibody to a polypeptide of interest (eg, the antibody is a detectable substrate (eg, the antibody is a fluorescent compound, enzyme substrate, radioactive compound or The second antibody, which can be conjugated with the luminescent compound) or which recognizes the first antibody, comprises means for detecting) which can be conjugated with a detectable substrate.

  In another specific embodiment of the invention, the kit is a diagnostic kit for use in screening sera containing antibodies specific for proliferative and / or cancerous polynucleotides and polypeptides. . Such a kit may comprise a control antibody that does not react with the polypeptide of interest. Such a kit can comprise a substantially isolated polypeptide antigen comprising an epitope that is specifically immunoreactive with at least one anti-polypeptide antigen antibody. Further, such kits are for detecting binding of the antibody to an antigen (eg, the antibody can be conjugated with a fluorescent compound such as fluorescein or rhodamine, which can be detected by flow cytometry). Means. In certain embodiments, the kit can comprise a recombinantly produced polypeptide antigen or a chemically synthesized polypeptide antigen. The polypeptide antigens of the kit can also be attached to a solid support.

  In a more specific embodiment, the detection means of the above kit comprises a solid support to which the polypeptide antigen is attached. Such a kit also comprises a non-attached reporter labeled anti-human antibody. In this embodiment, binding of the antibody to the polypeptide antigen can be detected by binding of the reporter labeled antibody.

  In a further embodiment, the present invention includes a diagnostic kit for use in screening serum containing an antigen of a polypeptide of the present invention. The diagnostic kit comprises a substantially isolated antibody that is specifically immunoreactive with a polypeptide or polynucleotide antigen, and means for detecting binding of the polynucleotide or polypeptide antigen to the antibody. . In one embodiment, the antibody is attached to a solid support. In certain embodiments, the antibody can be a monoclonal antibody. The detection means of the kit can include a second labeled monoclonal antibody. Alternatively or additionally, the detection means may include a labeled, competing antigen.

  In one diagnostic configuration, the test serum reacts with a solid phase reagent having a surface-bound antigen obtained by the method of the present invention. Depending on the amount of anti-antigen antibody bound on the solid support, after binding the specific antigen antibody to this reagent and removing unbound serum components by washing, to bind the reporter to this reagent, This reagent is reacted with a reporter-labeled anti-human antibody. The reagent is washed again to remove unbound labeled antibody and the amount of reporter associated with the reagent is determined. Typically, a reporter is an enzyme that is detected by incubating this solid phase in the presence of a suitable fluorometric, luminescent or colorimetric substrate (Sigma, St. Louis, MO).

  The solid surface reagent in the above assay is prepared by known techniques for attaching protein material to a solid support material (eg, polymeric beads, dipstick, 96 well plate or filtration material). These attachment methods generally include non-specific adsorption of proteins to the support or chemically active groups (eg, activated carboxyl groups, hydroxyl groups, or aldehyde groups) on the solid support. Covalent attachment (typically via a free amine group). Alternatively, streptavidin-coated plates can be used with biotinylated antigens.

  Thus, the present invention provides an assay system or kit for performing this diagnostic method. The kit generally comprises a support having a surface-bound recombinant antigen and a reporter-labeled anti-human antibody for detecting the surface-bound anti-antigen antibody.

(Albumin fusion protein)
The present invention relates generally to albumin fusion proteins and methods for treating, interfering with or ameliorating a disease or disorder. As used herein, “albumin fusion protein” refers to the fusion of at least one albumin molecule (or fragment or variant thereof) to at least one therapeutic protein (or fragment or variant thereof). A protein that is formed. The albumin fusion protein of the present invention comprises at least a fragment or variant of a therapeutic protein and at least a fragment or variant of human serum albumin, preferably by gene fusion (ie, the albumin fusion protein comprises all or part of the therapeutic protein). The encoding polynucleotide is linked with or linked to another one (generated by translation of the nucleic acid bound in the backbone to the polynucleotide encoding all or part of albumin). Once the therapeutic protein and albumin protein are part of the albumin fusion protein, each may be referred to as a “part”, “region” or “component” of the albumin fusion protein.

  In preferred embodiments, the present invention provides albumin fusion proteins encoded by the polynucleotides or albumin fusion constructs described in Table 1 or Table 2. Polynucleotides encoding these albumin fusion proteins are also encompassed by the present invention.

  Preferred albumin fusion proteins of the invention include, but are not limited to, albumin fusion proteins encoded by: at least one poly that encodes at least one molecule of a therapeutic protein (or fragment or variant thereof). Nucleic acid molecules comprising or consisting of a polynucleotide encoding at least one molecule of albumin (or a fragment or variant thereof) linked in-frame to a nucleotide; described in Table 1, Table 2 or Examples Albumin (or fragment or modified thereof) linked in-frame to at least one polynucleotide encoding at least one molecule of the therapeutic protein (or fragment or variant thereof) A nucleic acid molecule comprising or consisting of a polynucleotide encoding at least one molecule; or at least a therapeutic protein (or fragment or variant thereof) further comprising, for example, one or more of the following elements: A nucleic acid molecule comprising or consisting of a polynucleotide encoding at least one molecule of albumin (or a fragment or variant thereof) linked in frame to at least one polynucleotide encoding one molecule: (1) Functional self-replicating vectors (eg, including but not limited to shuttle vectors, expression vectors, integration vectors and / or replication systems), (2) regions for transcription initiation (eg, promoter regions (eg, regulatory) Possible promoter Or inducible promoters, constitutive promoters)), (3) a region for transcription termination, (4) a leader sequence, and (5) a selectable marker.

  In one embodiment, the invention provides an albumin fusion protein comprising or consisting of a therapeutic protein (eg, as described in Table 1) and a serum albumin protein. In other embodiments, the present invention provides albumin fusion proteins comprising or consisting of biologically active fragments and / or therapeutically active fragments of therapeutic proteins and serum albumin proteins. In other embodiments, the invention provides albumin fusion proteins comprising or consisting of biologically active variants and / or therapeutically active variants of therapeutic proteins and serum albumin proteins. In a preferred embodiment, the serum albumin protein component of the albumin fusion protein is a mature portion of serum albumin.

  In further embodiments, the present invention provides albumin fusion proteins comprising or consisting of therapeutic proteins and biologically active fragments and / or therapeutically active fragments of serum albumin proteins. In further embodiments, the present invention provides albumin fusion proteins comprising or consisting of therapeutic proteins and biologically active variants and / or therapeutically active variants of serum albumin proteins. In preferred embodiments, the therapeutic protein portion of the albumin fusion protein is the mature portion of the therapeutic protein.

  In further embodiments, the present invention provides biologically active fragments and variants and / or therapeutically active fragments or variants of therapeutic proteins, and biologically active fragments or variants of serum albumin proteins. And / or an albumin fusion protein comprising or consisting of a therapeutically active fragment or variant. In a preferred embodiment, the present invention provides an albumin fusion protein comprising or consisting of a mature portion of a therapeutic protein and a mature portion of serum albumin.

  Preferably, the albumin fusion protein comprises HA as the N-terminal portion and the therapeutic protein as the C-terminal portion. Alternatively, an albumin fusion protein comprising a therapeutic protein as the N-terminal portion and HA as the C-terminal portion can also be used.

  In other embodiments, the albumin fusion protein has a therapeutic protein fused to both the N-terminus and C-terminus of albumin. In a preferred embodiment, the therapeutic protein fused at the N-terminus and the therapeutic protein fused at the C-terminus are the same therapeutic protein. In an alternative preferred embodiment, the therapeutic protein fused at the N-terminus and the therapeutic protein fused at the C-terminus are different therapeutic proteins. In another preferred embodiment, the therapeutic protein fused at the N-terminus and the therapeutic protein fused at the C-terminus are the same or related diseases, disorders or conditions (eg, in the “Preferred indication Y” column of Table 1). Different therapeutic proteins that can be used to treat or prevent. In another preferred embodiment, the therapeutic protein fused at the N-terminus and the therapeutic protein fused at the C-terminus generally occur simultaneously, simultaneously or continuously in the patient, or In general, treating, reducing or preventing a disease or disorder known in the art to occur in relation to each other in a patient (such as those listed in the “Preferred indications Y” column of Table 1). Different therapeutic proteins that can be used to

  Exemplary fusion proteins of the invention comprising a plurality of therapeutic proteins fused at the N-terminus of albumin and therapeutic proteins fused at the C-terminus include, but are not limited to: GCSF-HSA-EPO, EPO-HSA-GCSF, IFNα-HSA-IL2, IL2-HSA-IFNα, GCSF-HSA-IL2, IL2-HSA-GCSF, IL2-HSA-EPO, EPO-HSA-IL2, IL3-HSA-EPO, EPO- HSA-IL3, GCSF-HSA-GMCSF, GMCSF-HSA-GCSF, IL2-HSA-GMCSF, GMCSF-HSA-IL2, PTH-HSA-calcitonin, calcitonin-HSA-PTH, PTH-PTH-HSA-calcitonin, calcitonin HSA-PTH -PTH, PTH-calcitonin-HSA-PTH, or PTH-HSA-calcitonin-PTH.

  The albumin fusion protein of the present invention comprises a predetermined therapeutic protein X or a variant thereof fused to the N-terminus or C-terminus of the albumin fusion protein of the present invention and / or the N-terminus or C-terminus of albumin or a variant thereof. Includes proteins comprising one, two, three, four or more molecules. A molecule of a given therapeutic protein X or variant thereof can be in any number of directions (including but not limited to: “head to head” direction (eg, the N-terminus of one molecule of therapeutic protein X is , When fused to the N-terminus of another molecule of therapeutic protein X) or in the “head-to-tail” direction (eg, the C-terminus of one molecule of therapeutic protein X is fused to the N-terminus of another molecule of therapeutic protein X). ))).

  In one embodiment, one, two, three or more of the tandemly oriented therapeutic protein X polypeptides (or fragments or variants thereof) are N-terminal or C-terminal of the albumin fusion protein of the invention, and / or Alternatively, it is fused to the N-terminus and / or C-terminus of albumin or a variant thereof.

  In certain embodiments, one, two, three, four, five or more of the tandemly oriented PTH molecules are fused to the N-terminus or C-terminus of albumin or a variant thereof. For example, one, two, three, four, five or more of tandemly oriented PTH molecules (including but not limited to PTH molecules comprising or consisting of amino acids 1-34) , Fused to the N-terminus or C-terminus of albumin or a variant thereof. Exemplary fusion proteins of the invention comprising multiple protein portions of PTH include, but are not limited to: PTH-PTH-HSA, HSA-PTH-PTH, PTH-PTH-PTH-HSA, HSA -PTH-PTH-PTH, PTH-PTH-PTH-PTH-HSA or HSA-PTH-PTH-PTH-PTH.

  In another specific embodiment, one, two, three, four, five or more of the tandemly oriented GLP-1 molecules are fused to the N-terminus or C-terminus of albumin or a variant thereof. . For example, one, two, three, four, five or more of tandemly oriented GLP-1 molecules (GLP-1 molecules comprising or consisting of amino acids 7-36 (residue 8 is alanine). (Eg, but not limited to) (for example, US Pat. No. 5,545,618, which is incorporated herein by reference in its entirety). Are fused to the N-terminus or C-terminus of albumin or variants thereof. Exemplary fusion proteins of the invention comprising multiple protein portions of GLP-1 include, but are not limited to: GLP1-GLP1-HSA, HSA-GLP1-GLP1, GLP1 variant-GLP1 variant HSA, HSA-GLP1 variant-GLP1 variant, GLP1 variant-GLP1-HSA, HSA-GLP1 variant-GLP1, GLP1-GLP1 variant-HSA or HSA-GLP1-GLP1 variant. Particularly preferred embodiments are GLP-1 tandem fusions such as construct ID number 3070 and proteins encoded by such constructs.

  The albumin fusion protein of the present invention comprises a predetermined therapeutic protein X or a variant thereof fused to the N-terminus or C-terminus of the albumin fusion protein of the present invention and / or the N-terminus or C-terminus of albumin or a variant thereof. Further encompassed are proteins comprising one, two, three, four or more molecules, wherein these molecules are linked via a peptide linker. Examples include the peptide linkers described in US Pat. No. 5,073,627 (incorporated herein by reference). Albumin fusion proteins comprising multiple therapeutic protein X polypeptides separated by peptide linkers can be generated using conventional recombinant DNA techniques. Linkers are particularly important when fusing small peptides to large HSA molecules. The peptide itself can be a linker by fusing tandem copies of the peptide (see, eg, GLP-1), or other known linkers can be used. Constructs incorporating the linker are listed in Table 2 or will be apparent from testing SEQ ID NO: Y.

  Furthermore, the albumin fusion proteins of the present invention also provide therapeutic protein X at the N-terminus and / or C-terminus of albumin or a variant thereof in a manner that allows the formation of intramolecular and / or intermolecular multimeric forms. Or it can be produced by fusing variants thereof. In one embodiment of the invention, the albumin fusion protein may be in monomeric or multimeric form (ie, dimer, trimer, tetramer and higher order multimer). In a further embodiment of the invention, the therapeutic protein portion of the albumin fusion protein can be in monomeric or multimeric form (ie, dimer, trimer, tetramer and higher order multimer). In certain embodiments, the therapeutic protein portion of the albumin fusion protein is in multimeric form (ie, dimer, trimer, tetramer, and higher order multimer), and the albumin protein portion is in monomeric form.

  In addition to albumin fusion proteins in which the albumin moiety is fused to the N-terminus and / or C-terminus of the therapeutic protein portion, the albumin fusion proteins of the present invention may also comprise a therapeutic protein or peptide of interest (eg, as disclosed in Table 1). A therapeutic protein X, or an antibody or fragment or variant thereof that binds to a therapeutic protein) can be generated by inserting into the internal region of the HA. For example, in the protein sequence of the HA molecule, a number of loops or turns exist between the end point and the start point of the α helix, which are stabilized by disulfide bonds. The loops (PDB identifiers 1AO6, 1BJ5, 1BKE, 1BM0, 1E7E-1E7I and 1UOR) as determined from the crystal structure of HA are mostly extended away from the molecular body. These loops are useful for the insertion or internal fusion of therapeutically active peptides (especially those that require secondary structure to be functional) or therapeutic proteins and have specific biological activities Essentially produces albumin molecules.

  Loops in the human albumin structure into which peptides or polypeptides can be inserted to produce albumin fusion proteins of the invention include: Val54 to Asn61, Thr76 to Asp89, Ala92 to Glu100, Gln170 to Ala176, His247 to Glu252. , Glu266-Glu277, Glu280-His288, Ala362-Glu368, Lys439-Pro447, Val462-Lys475, Thr478-Pro486 and Lys560-Thr566. In a more preferred embodiment, the peptide or polypeptide is inserted into the Val54-Asn61 loop, Gln170-Ala176 loop and / or Lys560-Thr566 loop of mature human albumin (SEQ ID NO: 1038).

  The peptide to be inserted can be derived from either a phage display library or a synthetic peptide library screened for a particular biological activity, or from an active portion of a molecule having the desired function. Furthermore, a random peptide library can be generated within a specific loop of the HA molecule or can be generated by inserting a randomized peptide into a specific loop of the HA molecule, where all possible amino acids Combinations are presented.

Such libraries can be generated for HA or HA domain fragments by one of the following methods:
Randomized mutations of amino acids within one or more peptide loops within a HA or HA domain fragment. Any one, more or all residues in the loop can be mutated in this manner.

Substitution of one or more loops of HA or HA domain fragments or HA or HA domain fragments of a randomized peptide of length X _n, where X is an amino acid and n is the number of residues Insertion into one or more loops (ie, internal fusion);
N-terminal, C-terminal, or N-terminal and C-terminal peptide / protein fusions in addition to (a) and / or (b).

  HA or HA domain fragments can also be rendered multifunctional by grafting peptides from different screens of different loops against different targets to the same HA or HA domain fragment.

  In a preferred embodiment, the peptide inserted into the loop of human serum albumin is a peptide fragment or peptide variant of a therapeutic protein disclosed in Table 1. More specifically, the present invention relates to at least 7, at least 8 amino acids, at least 9 amino acids, at least 10 amino acids, at least 11 amino acids, at least 12 amino acids, at least 13 inserted in a loop of human serum albumin. An albumin fusion protein comprising an amino acid length, at least 14 amino acids, at least 15 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids or at least 40 amino acids Include. The present invention also includes at least 7, at least 8 amino acids, at least 9 amino acids, at least 10 amino acids, at least 11 amino acids, at least 12 amino acids, at least 13 amino acids, at least 14 fused to the N-terminus of human serum albumin. Albumin fusion proteins comprising peptide fragments or peptide variants of amino acid length, at least 15 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids or at least 40 amino acids in length. The present invention also includes at least 7, at least 8 amino acids, at least 9 amino acids, at least 10 amino acids, at least 11 amino acids, at least 12 amino acids, at least 13 amino acids, at least 14 fused to the C-terminus of human serum albumin. Albumin fusion proteins comprising peptide fragments or peptide variants of amino acid length, at least 15 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids or at least 40 amino acids in length. For example, a short peptide described in Table 1 and Table 2 (eg, therapeutic agent Y) can be inserted into the albumin loop.

  In general, an albumin fusion protein of the invention may have one HA-derived region and one therapeutic protein-derived region. However, multiple regions of each protein can be used to make an albumin fusion protein of the invention. Similarly, more than one therapeutic protein can be used to make an albumin fusion protein of the invention. For example, a therapeutic protein can be fused to both the N-terminus and C-terminus of HA. In such a configuration, the therapeutic protein moieties may be the same therapeutic protein molecule or different therapeutic protein molecules. The structure of the dual functional albumin fusion protein can be shown as X-HA-Y or Y-HA-X.

For example, anti-BLys ^™ scFv-HA-IFNα-2b fusions can be prepared to modulate an immune response against IFNα-2b by anti-BLys ^™ scFv. An alternative is to use an HA fusion (eg, HA-IFNα-2b fusion mixed with HA-anti-BLys ^™ scFv fusion or other HA fusions in various ratios depending on function (such as half-life). ) To produce double (or even multiple) functional doses.

  A bifunctional or multifunctional albumin fusion protein can also be used at the opposite end of the HA to target the therapeutic protein portion of the fusion to the target organ or cell type via the protein or peptide. Can be prepared.

As an alternative to known therapeutic molecule fusions, these peptides are typically 6, 8, 12, 20 or 25 or X _n, where X is an amino acid (aa) and n is a residue A randomized amino acid), or a library constructed as a fusion to N-terminal, C-terminal, or N-terminal and C-terminal fusions of HA or HA domain fragments, Here, all possible combinations of amino acids were presented. A particular advantage of this approach is that the peptide on the HA molecule can be selected in situ, so that the properties of this peptide are similar to those for peptides derived by any other method and then bound to HA. It can be selected, perhaps not modified.

  Furthermore, the albumin fusion proteins of the present invention can include a linker peptide between the fusion moieties to provide greater physical separation between these moieties, thus making the therapeutic protein moiety accessible (eg, its cognate receptor). Can be maximized). The linker peptide can consist of amino acids so that it is flexible or more rigid.

  The linker sequence can be cleaved by a protease or chemically to yield a growth hormone-related moiety. Preferably, the protease is a protease naturally produced by the host (eg, S. cerevisiae protease kex2 or equivalent protease).

  Thus, as described above, the albumin fusion protein of the present invention may have the following formula: R1-L-R2; R2-L-R1 or R1-L-R2-L-R1, where R1 is A sequence of at least one therapeutic protein, peptide or polypeptide, but not necessarily the same therapeutic protein, L is a linker and R2 is a serum albumin sequence.

  In a preferred embodiment, an albumin fusion protein of the invention comprising a therapeutic protein has an increased shelf life compared to the shelf life of the same therapeutic protein when not fused to albumin. The effective period typically refers to the period during which the therapeutic activity of the therapeutic protein in solution or in some other stored formulation is stable without undue loss of therapeutic activity. Many therapeutic proteins are highly unstable in their unfused state. As described below, the typical shelf life of these therapeutic proteins is significantly extended when incorporated into the albumin fusion proteins of the present invention.

  An albumin fusion protein of the invention having a “prolonged” or “extended” shelf life exhibits a higher therapeutic activity compared to a standard subjected to the same storage and handling conditions. This standard can be an unfused full length therapeutic protein. If the therapeutic protein portion of the albumin fusion protein is an analog, if it is a variant, or otherwise modified, or if it does not include the complete sequence for that protein, the extension of the therapeutic activity is the analog , Variants, altered peptides or full sequence non-fused equivalents may alternatively be compared. By way of example, an albumin fusion protein of the invention has an activity that is greater than about 100% of the standard therapeutic activity when subjected to the same storage and handling conditions as the standard when compared at a given time point, or a standard treatment It may retain activity greater than about 105%, 110%, 120%, 130%, 150% or 200% of activity.

  The shelf life can also be evaluated for the therapeutic activity remaining after storage, normalized to the therapeutic activity at the beginning of storage. An albumin fusion protein of the invention having an extended shelf life, as demonstrated by prolonged therapeutic activity, is greater than about 50% of the therapeutic activity of an equivalent unfused therapeutic protein when subjected to the same conditions. It can retain high therapeutic activity, about 60%, 70%, 80%, 90% or more therapeutic activity. For example, as discussed in Example 38, an albumin fusion protein of the invention comprising hGH fused to a full-length HA sequence can be recovered in solution under various temperature conditions for a period of up to 5 weeks or more. It can retain about 80% or more of the activity.

(Fusion protein expression)
The albumin fusion proteins of the present invention can be produced as recombinant molecules by secretion from yeast, microorganisms such as bacteria, or human or animal cell lines. Preferably, the polypeptide is secreted from the host cell.

  Certain embodiments of the present invention include DNA constructs encoding signal sequences effective to direct secretion in yeast, particularly signal sequences derived from yeast (especially homologous to yeast hosts), and There is no yeast-derived prosequence between the signal polypeptide and the mature polypeptide, including the fusion molecule of the first aspect.

  The Saccharomyces cerevisiae invertase signal is a preferred example of a signal sequence derived from yeast.

  Conjugates of the type prepared by Poznansky et al. (FEBS Lett. 239: 18 (1988)), where separately prepared polypeptides are linked by chemical cross-linking, are not contemplated.

  The present invention also includes cells (preferably yeast cells) that have been transformed to express an albumin fusion protein of the present invention. In addition to the transformed host cells themselves, the present invention also provides for a culture of these cells (preferably a monoclonal (clonal homogeneous) culture or a culture derived from a monoclonal culture) in nutrient medium. Contemplate. If the polypeptide is secreted, the medium contains the polypeptide with the cells or without the cells if the cells have been removed by filtration or centrifugation. Many expression systems (including bacteria (eg, E. coli and Bacillus subtilis), yeast (eg, Saccharomyces cerevisiae, Kluyveromyces lactis and Pichia pastoris), filamentous fungi (eg, Aspergillus) and plant cells, animals Are known and can be used.

Preferred yeast strains to be used in the production of albumin fusion proteins are D88, DXY1 and BXP10. D88 [leu2-3, leu2-122, can1, pr1, ubc4] is a derivative of the parent strain AH22his ⁺ (also known as DB1; see, eg, Sleep et al., Biotechnology 8: 42-46 (1990)). . This strain contains a leu2 mutation that allows auxotrophic selection of a 2 micron based plasmid containing the LEU2 gene. D88 also shows inhibition of PRB1 in glucose excess conditions. The PRB1 promoter is normally controlled by two checkpoints that monitor glucose levels and growth stages. This promoter is activated in wild-type yeast upon glucose depletion and entry into the stationary phase. Strain D88 shows suppression by glucose but maintains induction upon entry into the stationary phase. The PRA1 gene encodes YscA endoprotease A, a yeast vacuolar protease, which is localized in the ER. The UBC4 gene is in the ubiquitination pathway and is involved in targeting short-lived and aberrant proteins for ubiquitin-dependent degradation. This isolation of the ubc4 mutation has been found to increase the copy number of the expression plasmid in the cell and increase the expression level of the desired protein expressed from the plasmid (eg, International Publication No. WO99 / 00504). (See, which is hereby incorporated by reference in its entirety)).

  DXY1, a derivative of D88, has the following genotype: [leu2-3, leu2-122, can1, pr1, ubc4, ura3 :: yap3]. In addition to the mutation isolated in D88, this strain also has a knockout of YAP3 protease. This protease primarily results in cleavage of dibasic residues (RR, RK, KR, KK), but can also facilitate cleavage at a single basic residue in the protein. Isolation of this yap3 mutation resulted in high levels of full-length HSA production (eg, US Pat. No. 5,965,386 and Kerry-Williams et al., Yeast 14: 161-169 (1998)) Is incorporated herein by reference).

  BXP10 has the following genotype: [leu2-3, leu2-122, can1, pr1, ubc4, ura3, yap3 :: URA3, lys2, hsp150 :: LYS2, pmt1 :: URA3]. In addition to the mutation isolated in DXY1, this strain also has knockouts of the PMT1 gene and the HSP150 gene. The PMT1 gene is a member of an evolutionarily conserved family of dolichyl-phosphate-D-mannose protein O-mannosyltransferase (Pmt). The transmembrane topology of Pmt1p suggests that it is an endoplasmic reticulum integral protein that has a role in O-linked glycosylation. This mutation serves to reduce / eliminate O-linked glycosylation of the HSA fusion (see, eg, International Publication No. WO 00/44772, which is hereby incorporated by reference in its entirety). ) Studies have shown that Hsp150 protein is poorly separated from rHA by ion exchange chromatography. Mutations in the HSP150 gene remove potential contaminants that have proven difficult to remove by standard purification techniques. See, for example, US Pat. No. 5,783,423, which is hereby incorporated by reference in its entirety.

The desired protein is produced in a conventional manner, for example from a coding sequence inserted into the host chromosome or from a coding sequence on a free plasmid. Yeast is transformed with the coding sequence of the desired protein in any conventional manner, such as electroporation.
Methods for transformation of yeast by electroporation are described by Becker and Guarente (1990) Methods Enzymol. 194,182.

  Successfully transformed cells (ie, cells that contain a DNA construct of the present invention) can be identified by well-known techniques. For example, cells resulting from the introduction of the expression construct can be grown to produce the desired polypeptide. The cells are harvested and lysed, and their DNA contents are determined by Southern (1975) J. MoI. Mol. Biol. 98, 503 or Berent et al. (1985) Biotech. 3, 208 can be tested for the presence of DNA using methods such as those described by. Alternatively, the presence of the protein in the supernatant can be detected using an antibody.

  Useful yeast plasmid vectors include pRS403-406 and pRS413-416, which are described in Stratagene Cloning Systems. Generally available from La Jolla, CA 92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are yeast integration plasmids (YIp) and incorporate the yeast selectable markers HIS3, 7RP1, LEU2 and URA3. Plasmid pRS413-416 is a yeast chromosomal plasmid (Ycp).

  Preferred vectors for expressing albumin fusion proteins in yeast include pPPC0005, pScCHSA, pScNHSA and pC4: HSA, which are described in detail in Example 1. FIG. 2 shows a map of the pPPC0005 plasmid that can be used as a basic vector in which a polynucleotide encoding a therapeutic protein can be cloned to form an HA fusion. This is because PRB1 S.I. cerevisiae promoter (PRB1p), fusion leader sequence (FL), DNA encoding HA (rAH) and ADH1 S. cerevisiae. cerevisiae terminator sequence. The sequence of the fusion leader sequence is the first 19 amino acids of the human serum albumin signal peptide (SEQ ID NO: 1094) and the last 5 amino acids of the mating factor α1 promoter (SLDKR, which is incorporated herein by reference in its entirety, EP-A-387 319).

  Plasmids pPPC0005, pScCHSA, pScNHSA and pC4: HSA were deposited on April 11, 2001 at the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 2010, A -3276, PTA-3279 and PTA-3277 were given. Another vector useful for expression of albumin fusion proteins in yeast, the pSAC35 vector, is described in Sleep et al., BioTechnology 8:42 (1990), which is hereby incorporated in its entirety. Incorporated as a reference.

Another yeast promoter that can be used to express an albumin fusion protein is the MET25 promoter. See, for example, Dominik Mumburg, Rol Muller and Martin Funk, Nucleic Acids Research, 1994, Vol. 22, no. 25, pp. See 5767-5768.
The Met25 promoter is 383 bases long (bases -382 to -1) and the genes expressed by this promoter are also known as Met15, Met17 and YLR303W. A preferred embodiment uses the following sequence, where the Not1 site used for cloning is underlined at the 5 ′ end of the following sequence and the ATG start codon is underlined at the 3 ′ end: doing.

相補的な接着末端を介して、ＤＮＡをベクターに作動可能に連結するための種々の方法が、開発されている。例えば、相補的なホモポリマー区域が、ベクターＤＮＡに挿入されるべきＤＮＡセグメントに付加され得る。次いで、このベクターおよびＤＮＡセグメントは、相補的なホモポリマーテイル間での水素結合によって連結されて、組換えＤＮＡ分子を形成する。

Various methods have been developed for operably linking DNA to a vector via complementary cohesive ends. For example, complementary homopolymer sections can be added to the DNA segment to be inserted into the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between complementary homopolymer tails to form a recombinant DNA molecule.

Synthetic linkers containing one or more restriction sites provide an alternative way of linking DNA segments to vectors. The DNA segment produced by endonuclease restriction digestion may be bacteriophage T4 DNA polymerase or E. coli. E. coli DNA polymerase I (which removes overhanging gamma single-stranded ends by its 3′5 ′ exonucleolytic activity and fills indented 3 ′ ends by their polymerization activity (fill
in) an enzyme).

  Thus, the combination of these activities produces blunt-ended DNA segments. This blunt-ended segment is then incubated with a large molar excess of linker molecule in the presence of an enzyme (eg, bacteriophage T4 DNA ligase) that can catalyze the ligation of blunt-ended DNA molecules. Therefore, the reaction product is a DNA segment having a polymer linker sequence at its end. These DNA segments are then cleaved with appropriate restriction enzymes and ligated into an expression vector that has been cleaved with an enzyme that produces termini compatible with the termini of the DNA segment.

  Synthetic linkers containing various restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies Inc, New Haven, CT, USA.

  A desired method for modifying DNA according to the present invention is, for example, by using the polymerase chain reaction as disclosed by Saiki et al. (1988) Science 239, 487-491 when HA variants are prepared. is there. In this method, the DNA to be enzymatically amplified is flanked by two specific oligonucleotide primers that are incorporated into the amplified DNA itself. These specific primers can contain restriction endonuclease recognition sites that can be used to clone into the expression vector using methods known in the art.

  Exemplary genera of yeasts that are contemplated to be useful in the practice of the present invention as hosts for expressing albumin fusion proteins are: Pichia (Hansenula), Saccharomyces, Kluyveromyces, Candida, Toluropsis, Torulaspora, Schizosaccharomyces, Citeromyces, Pachysolen, Debaromyces, Metschunikowia, Rhodosporidium, Leucosporidium, Botryoascus, Sporidiobolus, Endosomis A preferred genus is a genus selected from the group consisting of Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia and Torulaspora. Saccharomyces spp. Examples of S. cerevisiae, S. et al. italicus and S. rouxii.

  Kluyveromyces spp. Examples of K.K. fragilis, K.M. lactis and K. et al. marxianus. Suitable Torulaspora species are T. et al. delbrueckii. Pichia (Hansenula) spp. An example of P.I. angusta (formerly H. polymorpha), P. a. anomala (formerly H. anomala) and P. anomala. pastoris. S. Methods for transformation of cerevisiae are generally taught in EP 251 744, EP 258 067 and WO 90/01063, all of which are hereby incorporated by reference.

  Preferred exemplary species of Saccharomyces include S. cerevisiae. cerevisiae, S. et al. italicus, S.M. and diastaticus and Zygosaccharomyces rouxii. Preferred exemplary species of Kluyveromyces include K. cerevisiae. fragilis and K.M. lactis. Preferred exemplary species of Hansenula include H. polymorpha (now Pichia angelsta), H. et al. anomala (now Pichia anomala) and Pichia capsules. Further preferred exemplary species of Pichia species include P. pastoris. Preferred exemplary species of Aspergillus include A. niger and A.M. nidulans. Preferred exemplary species of Yarrowia include Y. and lipolytica. Many preferred yeast species are available from the ATCC. For example, the following preferred yeast species are available from the ATCC and are useful in the expression of albumin fusion proteins: Saccharomyces cerevisiae Hansen, sexual generation BY4743 yap3 mutant (ATCC Accession No. 4022731); Saccharomyces cerevisiae Hansen, Sex generation strain BY4743 hsp150 variant (ATCC accession number 4021266); Saccharomyces cerevisiae Hansen, sexual generation strain BY4743 pmt1 variant (ATCC accession number 4023792); Saccharomyces cerevisiae generation 44 62955 ; Saccharomyces diastaticus Andrews et Gilliland ex van der Walt, sexual generation (ATCC Accession No. 62987); Kluyveromyces lactis (Dombrowski) van der Walt, sexual generation (ATCC Accession No. 76492); Pichia angusta (Teunisson et al.) Kurtzman, Hansenula polymorpha Sexual generation deposited as de Morais et Mia, Sexual generation (ATCC accession number 26012); Aspergillus niger van Tieghem, Asexual generation (ATCC accession number 9029); Aspergillus niger van Tiechem, Asexual generation A CC Accession No. 16404); Aspergillus nidulans (Eidam) Winter, asexual generation (ATCC Accession No. 48756); and Yarrowia lipolytica (Wickerham, et al.) Van der Walt et von Arx, sexual generation (ATCC Accession No. 201847).

S. Suitable promoters for cerevisiae include PGKI gene, GAL1 gene, or GAL10 gene, CYCI, PHO5, TRPI, ADHI, ADH2, gene for glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase , Phosphofructokinase, triosephosphate isomerase, glucokinase, α-mating factor pheromone, [mating factor pheromone], PRBI promoter, GUT2 promoter, GPDI promoter, and part of the 5 ′ regulatory region of other promoters or upstream active sites And those related to a hybrid promoter (for example, the promoter of EP-A-258067) relating to a hybrid of a part of the 5 ′ regulatory region having The

  A convenient regulatable promoter for use in Schizosaccharomyces pombe is described in Maundrell (1990) J. MoI. Biol. Chem. 265, 10857-10864, a thiamine-repressed promoter from the nmt gene and a glucose-repressed jbpl gene promoter as described by Hoffman & Winston (1990) Genetics 124, 807-816.

  Methods for transforming Pichia for expression of heterologous genes are described, for example, by Cregg et al. (1993) and various Phillip patents (eg, US Pat. No. 4,857,467, incorporated herein by reference). ) And Pichia expression kits are described in Invitrogen BV, Leek, Netherlands and Invitrogen Corp. , San Diego, California. Suitable promoters include AOXI and AOX2. Gleeson et al. Gen. Microbiol. 132, 3459-3465 contain information on Hansenula vectors and transformation, suitable promoters are MOX1 and FMD1; whereas other publications from EP 361 991, Freeer et al. (1991) and Rhone-Poulenc Roller are: Kluyveromyces spp. Teaches how to express heterologous proteins in, and a suitable promoter is PGKI.

The transcription termination signal is preferably the 3 ′ flanking sequence of a eukaryotic gene containing appropriate signals for transcription termination and polyadenylation. Suitable 3 ′ flanking sequences can be, for example, sequences of genes that are naturally linked to the expression control sequences used (ie, can represent promoters). Alternatively, they are It may be different if the termination sequence of the cerevisiae ADHI gene is preferred.

The desired albumin fusion protein can be initially expressed with a secretory leader sequence that can be any leader effective in the selected yeast. Useful leaders in yeast are:
MPIF-1 signal sequence (eg, amino acids 1 to 21 of GenBank trust number AAB51134) MKVSVAALSCLMLVTALGSQA (SEQ ID NO: 2132)
Stanniocalcin signal sequence (MLQNSAVLLLLVISASA, SEQ ID NO: 1054)
The pre-pro region of the HSA signal sequence (eg, MKWVTFISLLFLSSAYSRRGVFRR, SEQ ID NO: 1176)
A pre-region of the HSA signal sequence (eg, MKWVTFISLLFLSFSS, SEQ ID NO: 1177) or a variant thereof (eg, MKWVSFISLLFLSFSYS) (SEQ ID NO: 1168)
Invertase signal sequence (eg, MLLQAFLFLLAGFAAKISA, SEQ ID NO: 1108)
Yeast mating factor α signal sequence (eg, MRFPSIFTAVLAFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDDFDVAVLPFSTNSTNGGLLFINTTIASIAKAEGTGNDFNGTSNTAQNGTDNGTD
K. lactis killer toxin leader sequence hybrid signal sequence (eg, MKWVSFISLLFLSSASRSRLEKR, SEQ ID NO: 1110)
HSA / MFα-1 hybrid signal sequence (also known as HSA / kex2) (eg, MKWVSFISLLFFSSAYSRSLDKR, SEQ ID NO: 1111)
K. lactis killer / MFα-1 fusion leader sequence (eg, MNIFYIFLFLLSFVQGSLDKR, SEQ ID NO: 1169)
Immunoglobulin Ig signal sequence (eg, MGWSCIILFLVATATGVHS, SEQ ID NO: 1095)
Fibulin B precursor signal sequence (eg, MERAAPSRRRVPLPLLLLGGLALLAAGVDA, SEQ ID NO: 1096)
Cluster precursor signal sequence (eg MMKTLLLFVGLLLTWESGQVLG, SEQ ID NO: 1097)
Insulin-like growth factor binding protein 4 signal sequence (eg, MLPLCLVAALLLAAGPGPSLG, SEQ ID NO: 1098)
A variant of the pre-pro-pre region of the HSA signal sequence (eg, MKVSVSISLFLFFSSAYSRRGVFRR (SEQ ID NO: 1167)),
MKWVTFISLLFLFAGVLG (SEQ ID NO: 1099),
MKWVTFISLLFLFSGVLG (SEQ ID NO: 1100),
MKWVTFISLLFLFGGVLG (SEQ ID NO: 1101),
Modified HSA leader HSA # 64
MKWVTFISLLFLFAGVSG (SEQ ID NO: 2133);
Modified HSA leader HSA # 66
MKWVTFISLLFLFGGVSG (SEQ ID NO: 2134);
Modified HSA (A14) leader
MKWVTFISLLFLFAGVSG (SEQ ID NO: 1102);
Modified HSA (S14) leader (also known as modified HSA # 65)-MKWVTFISLLFLFSGVSG (SEQ ID NO: 1103)
Modified HSA (G14) leader
MKWVTFISLLFLFGGVSG (SEQ ID NO: 1104), or MKWVTFISLLFLFGGVLGDLHKS (SEQ ID NO: 1105)
Consensus signal sequence (MPTWAWWLFLVLLLALWAPARG, SEQ ID NO: 1055)
Acid phosphatase (PH05) leader (eg, MFKSVVYSILAASLANA SEQ ID NO: 2135)
MFoz-1 pre-sequence 0 glucanase (BGL2) pre-sequence killer toxin leader killer toxin pre-sequence k. lactis killer toxin prepro (29 amino acids; pre 16 amino acids and pro 13 amino acids) MNIFYIFLFLLFSVQGLEHTHRRGSLDKR (SEQ ID NO: 2136)
S. diastaticus glucoalnylase I1 secretory leader sequence S. cerevisiae The hybrid leader gp67 signal sequence disclosed in the callsbergensis α-galactoside (MEL1) secretory leader sequence Candida glucoarnylase leader sequence EP-A-387 319 (incorporated herein by reference) (in relation to the baculovirus expression system) (eg GenBank trust number AAA72759 amino acids 1-19) or the natural leader of therapeutic protein X;
JP 62-096086 (approved as 911036516, incorporated herein by reference), S.P. cerevisiae invertase (SUC2) leader; or inulinase-MKLAYSLLPLAGVSAVINYKR (SEQ ID NO: 2137).
Modified TA57 Propeptide Leader Variant # 1-MKLKTVRSAVLSSSLFASQVLGQPIDDTESQTTSVNLMADDTESAFAATQTNSGLDVVGLISMAKR (SEQ ID NO: 2128)
Modified TA57 Propeptide Leader Variant # 2-MKLKTVRSAVLSSSLFASQVLGQPIDDTESQTTSVNLMADDTESAFAATQTNSGGLVVGLISMAEEGEEPKR (SEQ ID NO: 2129)
(Further methods for recombinant and synthetic production of albumin fusion proteins)
The present invention also relates to vectors, host cells, and production of albumin fusion proteins by synthetic and recombinant techniques comprising a polynucleotide encoding an albumin fusion protein of the invention. For example, the vector can be a phage vector, a plasmid vector, a viral vector, or a retroviral vector. Retroviral vectors can be replication competent or replication defective. In the latter case, virus propagation generally occurs only in complementing host cells.

  A polynucleotide encoding an albumin fusion protein of the invention can be linked to a vector containing a selectable marker for growth in a host. In general, plasmid vectors are introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, the vector can be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.

  The polynucleotide insert may contain any suitable promoter (eg, phage λPL promoter, E. coli lac promoter, trp promoter, phoA promoter and tac promoter, SV40 early promoter and SV40 late promoter, and promoter of retroviral LTR, to name a few. ) Should be operatively coupled to. Other suitable promoters are known to those skilled in the art. The expression construct further includes a site for transcription initiation, transcription termination, and a ribosome binding site for translation in the transcription region. The coding portion of the transcript expressed by this construct preferably comprises an initial translation start codon and a termination codon (UAA, UGA or UAG) appropriately located at the end of the polypeptide to be translated.

As indicated, the expression vector preferably includes at least one selectable marker.
Such markers include dihydrofolate reductase, G418, glutamine synthase or neomycin resistance genes for eukaryotic cell culture, and E. coli. Contains tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of suitable hosts are bacterial cells (eg, E. coli, Streptomyces and Salmonella typhimurium cells); fungal cells (eg, yeast cells (eg, Saccharomyces cerevisiae or Pichia pastoris (ATCC accession number 201178)) Insect cells (eg, Drosophila S2 and Spodoptera Sf9 cells); animal cells (eg, CHO cells, COS cells, NSO cells, 293 cells, and Bowes melanoma cells); and plant cells, but are not limited to these. Appropriate culture media and conditions for the above-described host cells are known in the art.

  Among the preferred vectors for use in bacteria are pQE70, pQE60 and pQE-9 (available from QIAGEN, Inc.); pBluescript vector, Pagescript vector, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene Cloning Systems, And ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (available from Pharmacia Biotech, Inc.). Among the preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG (available from Stratagene); and pSVK3, pBPV, pMSG and pSVL (available from Pharmacia). Preferred expression vectors for use in yeast systems include pYES2, pYD1, pTEF1 / Zeo, pYES2 / GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K. , And PAO815 (all available from Invitrogen, Carlbad, Calif.). Other suitable vectors will be readily apparent to those skilled in the art.

In one embodiment, a polynucleotide encoding an albumin fusion protein of the invention directs localization of the protein of the invention to a specific fraction of prokaryotic or eukaryotic cells and / or prokaryotic cells. Alternatively, it can be fused to a signal sequence that directs secretion of a protein of the invention from a eukaryotic cell. For example, E.I. In E. coli, it may be desirable to direct protein expression to the periplasmic space. Examples of signal sequences or proteins (fragments thereof) to which the albumin fusion proteins of the invention can be fused to direct polypeptide expression into the bacterial periplasmic space include pelB signal sequences, maltose binding protein (MBP) signals Sequence, MBP, ompA signal sequence, periplasm Examples include, but are not limited to, the signal sequence of the E. coli heat-labile enterotoxin B subunit and the signal sequence of alkaline phosphatase. Several vectors are commercially available for the construction of fusion proteins that direct the localization of proteins (pMAL species of vectors available from New England Biolabs (specific pMAL-p species)) in certain embodiments. The polynucleotide albumin fusion proteins of the invention can be fused to the pelB pectate lyase signal sequence, increasing the efficiency of expression and purification of such polypeptides in Gram negative bacteria.
See US Pat. Nos. 5,576,195 and 5,846,818, the contents of which are hereby incorporated by reference in their entirety.

Examples of signal peptides that can be fused to an albumin fusion protein of the invention to direct its secretion in mammalian cells include, but are not limited to:
a) MPIF-1 signal sequence (eg amino acids 1-21 of GenBank trust number AAB51134) MKVSVAALSCLMLVTALGSQA (SEQ ID NO: 2132)
b) Stanniocalcin signal sequence (MLQNSAVLLLLVISASA, SEQ ID NO: 1054)
c) Pre-pro region of HSA signal sequence (eg MKWVTFISLLFLSSAYSRRGVFRR, SEQ ID NO: 1176)
d) Pre-regions of the HSA signal sequence (eg MKWVTFISLLFLSFS, SEQ ID NO: 1177) or variants thereof, eg MKWVSFISLLFLSFS, (SEQ ID NO: 1168)
e) Invertase signal sequence (eg, MLLQAFLFLLAGFAAKISA, SEQ ID NO: 1108)
f) Yeast mating factor alpha signal sequence (eg MRFPSIFTAVFASSALAVNTTTEDETAQIPAEAVIGYSDLEGDDFDVAVLPFSNSTNNGLLFINTTINGASVNDSNGTDNGSDFATNT
g) K.K. lactis killer toxin leader sequence h) hybrid signal sequence (eg, MKWVSFISLFLFSSAYSRSLEKR, SEQ ID NO: 1110)
i) HSA / MFα-1 hybrid signal sequence (also known as HSA / kex2) (eg, MKWVSFISLFLFSSAYSRSLDKR, SEQ ID NO: 1111) j) lactis killer / MFα-1 fusion leader sequence (eg, MNIFYIFLFLLSFVQGSLDKR, SEQ ID NO: 1169)
k) Immunoglobulin Ig signal sequence (eg, MGWSCIILFLVATATGVHS, SEQ ID NO: 1095)
l) Fibulin B precursor signal sequence (eg, MERAAPSRRRVPLPLLLLGGLALLAAGVDA, SEQ ID NO: 1096)
m) crusterin precursor signal sequence (eg MMKTLLLFVGLLLTWESGQVLG, SEQ ID NO: 1097)
n) Insulin-like growth factor binding protein 4 signal sequence (eg MLPLCLVAALLLAAGPGPSLG, SEQ ID NO: 1098)
o) a pre-pro-region variant of the HSA signal sequence such as MKVSVSISLFLFFSSAYSRRGVFRR (SEQ ID NO: 1167), such as:
MKWVTFISLLFLFAGVLG (SEQ ID NO: 1099),
MKWVTFISLLFLFSGVLG (SEQ ID NO: 1100),
MKWVTFISLLFLFGGVLG (SEQ ID NO: 1101),
Modified HSA leader HSA # 64
MKWVTFISLLFLFAGVSG (SEQ ID NO: 2133);
Modified HSA leader HSA # 66
MKWVTFISLLFLFGGVSG (SEQ ID NO: 2134);
Modified HSA (A14) leader
MKWVTFISLLFLFAGVSG (SEQ ID NO: 1102);
Modified HSA (S14) leader (also known as modified HSA # 65)-MKWVTFISLLFLFSGVSG (SEQ ID NO: 1103),
Modified HSA (G14) leader
MKWVTFISLLFLFGGVSG (SEQ ID NO: 1104), or MKWVTFISLLFLFGGVLGDLHKS (SEQ ID NO: 1105)
p) Consensus signal sequence (MPTWAWWWLFLVLLLALWAPARG, SEQ ID NO: 1055)
q) Acid phosphatase (PH05) leader (eg MFKSVVYSILAASLANA, SEQ ID NO: 2135)
r) Pre-sequence of MFoz-1 s) Pre-sequence of 0 glucanase (BGL2) t) Killer toxin leader u) Pre-sequence of killer toxin v) k. lactis killer toxin prepro (29 amino acids; pre 16 amino acids and pro 13 amino acids) MNIFYIFLFLLFSVQGLEHTHRRGSLDKR (SEQ ID NO: 2136)
w) S.M. diastaticus glucoamylase II secretion leader sequence x) carlsbergensis α-galactosidase (MEL1) secretory leader sequence y) Candida glucoarnylase leader sequence z) hybrid leader aa) gp67 signal sequence (baculovirus expression system) disclosed in EP-A-387 319 (incorporated herein by reference) (E.g., amino acids 1-19 of GenBank trust number AAA72759) or bb) the natural leader X of therapeutic protein X;
cc) JP 62-096086 (approved as 911036516 and incorporated herein by reference), S. cerevisiae invertase (SUC2) leader; or dd) inulinase-MKLAYSLLLLPLAGVSAVINYKR (SEQ ID NO: 2137)
ee) Modified TA57 propeptide leader variant # 1-MKLKTVRSAVLSSSLFASQVLGQPIDDTESQTTSVNLLMADDTESAFATQNSGGLDVVGLISMAKR (SEQ ID NO: 2128).
ff) Modified TA57 Propeptide Leader Variant # 2-MKLKTVRSAVLSSSLFASQVLGQPIDDTESQTTSVNLLMADTEDAFATQNSGGLDVVGLISMAEEGEEPKR (SEQ ID NO: 2129)

Vectors that use glutamine synthase (GS) or DHFR as selectable markers can be amplified in the presence of drugs (methionine sulphoximine or methotrexate, respectively). An advantage of glutamine synthase-based vectors is that a glutamine synthase negative cell line (eg, a mouse myeloma cell line (NSO)) is available. Glutamine synthase expression systems can also function in glutamine synthase expressing cells (eg, Chinese hamster ovary (CHO) cells) by providing additional inhibitors to prevent endogenous gene function. The glutamine synthase expression system and its components are described in PCT Publication Nos. WO87 / 04462; WO86 / 05807; WO89 / 01036; WO89 / 10404; and WO91 / 06657 (all of these). Are incorporated herein by reference). In addition, glutamine synthase expression vectors are available from Lonza Biologics, Inc. (Portsmouth, NH). Expression and production of monoclonal antibodies in mouse myeloma cells using the GS expression system is described in Bebbington et al., Bio / technology 10: 169 (1992) and Biblia and Robinson Biotechnol. Prog. 11: 1 (1995) (incorporated herein by reference).

  The present invention also relates to host cells comprising the vector constructs described herein, and further includes host cells comprising the nucleotide sequences of the present invention. This nucleotide sequence is operably linked to one or more heterologous regulatory regions (eg, promoters and / or enhancers) using techniques known in the art. The host cell can be a higher eukaryotic cell such as a mammalian cell (eg, a human derived cell) or a lower eukaryotic cell such as a yeast cell, or the host cell can be a prokaryotic cell such as a bacterial cell. It can be a biological cell. A host strain that regulates the expression of the inserted gene sequence or a host strain that modifies or processes the gene product in the specific fashion desired can be selected. Expression from a particular promoter can be increased in the presence of a particular inducer; thus, expression of the engineered polypeptide can be controlled. Moreover, separate host cells have features and specific mechanisms for translation and post-translational protein processing and modification (eg, phosphorylation, cleavage). Appropriate cell lines can be chosen to ensure the desired modification and processing of the foreign protein expressed.

  Introduction of the nucleic acids and nucleic acid constructs of the present invention into host cells can be accomplished by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, or other methods. . Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the invention can be expressed by host cells that actually lack the recombinant vector.

In addition to including host cells containing the vector constructs discussed herein, the present invention also provides primary host cells, secondary host cells of vertebrate origin (particularly of mammalian origin). And immortalized host cells. These host cells have been engineered to delete or replace endogenous genetic material (eg, a coding sequence corresponding to a therapeutic protein can be replaced with an albumin fusion protein corresponding to the therapeutic protein), and And / or are designed to include genetic material (eg, heterologous polynucleotide sequences) (eg, include heterologous polynucleotide sequences such as the albumin fusion proteins of the invention corresponding to therapeutic proteins). Genetic material operably linked to the endogenous polynucleotide may activate, alter, and / or amplify the endogenous polynucleotide.
In addition, techniques known in the art include, via homologous recombination, heterologous polynucleotides (eg, polynucleotides encoding albumin protein, or fragments or variants thereof) and / or heterologous regulatory regions (eg, promoters and / or Or enhancers) can be used to operably link an endogenous polynucleotide sequence encoding a therapeutic protein (eg, US Pat. No. 5,641,670, issued 24 June 1997); International Publication No. WO 96/29411; International Publication No. WO 94/12650; Koller et al., Proc. Natl. Acad. Sci. USA 86: 8932-8935 (1989); 1989) And each of these disclosures is incorporated by reference in their entirety).

  The albumin fusion protein of the present invention comprises ammonium sulfate precipitation or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, hydrophobic charge It can be recovered from the recombinant cell culture and purified by well known methods including interaction chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is used for purification.

In a preferred embodiment, the albumin fusion protein of the invention is purified using anion exchange chromatography. As anion exchange chromatography, Q-Sepharose column, DEAE Sepharose column, Poros HQ column, Poros DEAE column, Toyopearl Q column, Toyopearl QAE column, Toyopearl DEAE column, Resource / Source QAE column and Resource / Source DEAE column , Fractogel Q columns, as well as Fractogel DEAE columns.
In certain embodiments, the albumin fusion proteins of the invention are purified using cation exchange chromatography. Cation exchange chromatography includes SP-Sepharose column, CM Sepharose column, Poros HS column, Poros CM column, Toyopearl SP column, Toyopearl CM column, Resource / Source S column and Resource / Source CM column, Fractogel S Columns and Fractogel CM columns and their equivalents and the like include, but are not limited to.

In certain embodiments, the albumin fusion proteins of the invention are purified using hydrophobic interaction chromatography. As hydrophobic interaction chromatography, phenyl-Sepharose column, butyl-Sepharose column, methyl-Sepharose column, octyl-Sepharose column, hexyl-Sepharose column, Porosphenyl column, Porosbutyl column, Porosmethyl column, Porosoctyl column, Poroshexyl column, Toyopearl phenyl column, Toyopearl butyl column, Toyopearl methyl column, Toyopearl octyl column, Toyopearl hexyl column, Resource / source phenyl column, Resource / source butyl column, Resource / source methyl column, Resource / source octyl column, Resource / source octyl column Hexyl column, Fractogel phenyl column, Fractog Butyl column, Fractogel methyl column, Fractogel octyl column, Fractogel hexyl column, as well as equivalents and analogues thereof, without limitation.
In certain embodiments, the albumin fusion proteins of the invention are purified using size exclusion chromatography. Size exclusion chromatography includes, but is not limited to, Sepharose S100 column, Sepharose S200 column, Sepharose S300 column, Sephadex resin column and equivalents and the like thereof.

  In certain embodiments, the albumin fusion proteins of the invention are purified using affinity chromatography. Affinity chromatography includes, but is not limited to, Mimic Dye affinity columns, peptide affinity columns, and antibody affinity columns. These columns are selective for either HSA or “fusion target” molecules.

  In preferred embodiments, the albumin fusion proteins of the invention are purified using one or more of the chromatographic methods listed above. In other preferred embodiments, the albumin fusion proteins of the invention are purified using one or more of the following chromatography columns: Q Sepharose FF column, SP Sepharose FF column, Q Sepharose high performance column, Blue Sepharose. FF column, blue column, phenyl sepharose FF column, DEAE sepharose FF column, or methyl column.

  Furthermore, the albumin fusion proteins of the present invention can be purified using the process described in PCT International Publication No. WO 00/44772, which is hereby incorporated by reference in its entirety. One skilled in the art can readily modify the process described therein for use in the purification of the albumin fusion protein of the invention.

  Albumin fusion proteins of the invention can be recovered from: products of chemical synthesis procedures; and products produced from recombinant or prokaryotic or eukaryotic hosts (eg, bacterial cells, yeast cells, higher plant cells). Insect cells, and mammalian cells). Depending on the host used in the recombinant production procedure, the polypeptides of the present invention may be glycosylated or non-glycosylated polypeptides. Furthermore, the albumin fusion proteins of the present invention may also include modification of the opening methionine residue (in some cases as a result of host-mediated processes). Thus, it is well known in the art that in all eukaryotic cells, the N-terminal methionine encoded by the translation initiation codon is generally efficiently removed from any protein after translation. N-terminal methionine in most proteins is also efficiently removed in most prokaryotes, but for some proteins this prokaryotic removal depends on the nature of the amino acid to which the N-terminal methionine is covalently linked. The process is inefficient.

In one embodiment, the yeast Pichia pastoris is used to express the albumin fusion protein of the invention in a eukaryotic cell system. Pichia pastoris is a methyltrophic yeast that can metabolize methanol as its sole carbon source. Major steps in methanol metabolic pathway is the oxidation of methanol to formaldehyde using O _2. This reaction is catalyzed by the enzyme alcohol oxidase. In order to metabolize methanol as its sole carbon source, Pichia pastoris is partially due to the relatively low affinity of alcohol oxidase for O _2, must generate high levels of alcohol oxidase. Consequently, in a growth medium that relies on methanol as the main carbon source, the promoter region of one of the two alcohol oxidase genes (AOX1) is very active. In the presence of methanol, alcohol oxidase produced from the AOX1 gene comprises up to approximately 30% of the total soluble protein in Pichia pastoris. Ellis, S.M. B. Et al., Mol. Cell. Biol. 5: 1111-21 (1985); Koutz, P .; J. et al. Yeast 5: 167-77 (1989); Tschop, J. et al. F. Et al., Nucl. Acids Res. 15: 3859-76 (1987). Accordingly, heterologous coding sequences (eg, polynucleotides of the present invention) under transcriptional control of all or part of the AOX1 regulatory sequences are expressed at very high levels in Pichia yeast grown in the presence of methanol.

  In one example, the plasmid vector pPIC9K is described in “Pichia Protocols: Methods in Molecular Biology”, D.M. R. Higgins and J.H. Express DNA encoding the albumin fusion protein of the present invention as shown herein in a Picea yeast system, essentially as described in the Cregg edition (The Humana Press, Totowa, NJ, 1998). Used for. This expression vector allows expression and secretion of the polypeptide of the present invention by virtue of the strong AOX1 promoter linked to a Pichia pastoris alkaline phosphatase (PHO) secretion signal peptide (ie, leader) located upstream of the multiple cloning site. to enable.

  Many other yeast vectors (eg, pYES2, pYD1, pTEF1 / Zeo, pYES2 / GS, pPICZ, pGAPZ, pGAPZα, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PAO815) As will be readily appreciated by those skilled in the art, as long as the proposed expression construct is required, as long as it provides appropriately positioned signals such as transcription, translation, secretion (if desired), including in-frame AUG Can be used instead of pPIC9K.

  In another embodiment, high level expression of a heterologous coding sequence (eg, such as a polynucleotide encoding an albumin fusion protein of the invention) causes the heterologous polynucleotide of the invention to be expressed in an expression vector (eg, such as pGAPZ or pGAPZα). And can be achieved by growing yeast cultures in the absence of methanol.

  Furthermore, the albumin fusion proteins of the present invention can be chemically synthesized using techniques known in the art (see, for example, Creighton, 1983, Proteins: Structures and Molecular Principles, WH Freeman & Co., NY). And Hunkapiller et al., Nature, 310: 105-111 (1984)). For example, a polypeptide corresponding to a fragment of the polypeptide can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence. Non-classical amino acids include, but are not limited to: D isomers of normal amino acids, 2,4-diaminobutyric acid, a-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, g -Abu, e-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-Butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoroamino acids, designer amino acids (eg, b-methyl amino acids), Ca-methyl amino acids, Na-methyl amino acids, and common amino acid analogs. Further, the amino acid can be D (dextrorotatory) or L (left-handed).

The invention can be used during or after translation, for example, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, antibody molecules or other cellular ligands. It includes an albumin fusion protein of the invention that is differentially modified, such as by binding. Any number of chemical modifications may be performed by known techniques including, but not limited to: specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH ₄ ; acetylation, Formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin;

  Additional post-translational modifications included by the present invention include, for example: N-linked or O-linked carbohydrate chains (N-terminal or C-terminal processing), attachment of chemical moieties to the amino acid backbone, N-linkage Chemical modification of type or O-linked carbohydrate chains and addition or deletion of N-terminal methionine residues as a result of prokaryotic host cell expression. The albumin fusion protein can also be modified with a detectable label (eg, an enzyme label, a fluorescent label, an isotope label or an affinity label) to allow detection and isolation of the protein.

Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; examples of luminescent materials include luminol; Examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive materials include iodine ( ¹²¹ I, ¹²³ I, ¹²⁵ I, ¹³¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ¹¹¹ In, ¹¹² In, ^{113 m} In, ^{115 m} In), technetium ( ⁹⁹ Tc, ^99m Tc), thallium ( ²⁰¹ Ti), gallium ( ⁶⁸ Ga, ⁶⁷ Ga), palladium ( ¹⁰³ Pd), molybdenum ( ⁹⁹ Mo), xenon ( ¹³³ Xe), fluorine ( ¹⁸ F), ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁵⁹ Gd, ¹⁴⁹ Pm, ¹⁴⁰ La, ¹⁷⁵ Yb, ¹⁶⁶ Ho, ⁹⁰ Y, ⁴⁷ Sc, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁴² Pr, ¹⁰⁵ Rh, and ⁹⁷ Ru.

In certain embodiments, an albumin fusion protein of the invention or fragment or variant thereof binds to a macrocyclic chelator that binds a radiometal ion, including but not limited to: ¹⁷⁷ Lu, ⁹⁰ Y, ¹⁶⁶ Ho , And ¹⁵³ Sm. This chelator is against the polypeptide. In a preferred embodiment, the radioactive metal ion that binds to the macrocyclic chelator is ¹¹¹ In. In another preferred embodiment, the radioactive metal ion associated with the macrocyclic chelator is ⁹⁰ Y. In certain embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N, N ′, N ″, N ′ ″-tetraacetic acid (DOTA). In other specific embodiments, DOTA binds to an antibody of the invention or fragment thereof via a linker molecule. Examples of linker molecules useful for DOTA binding to polypeptides are generally known in the art. See, for example, DeNardo et al., Clin Cancer Res. 4 (10): 2483-90 (1998); Peterson et al., Bioconjug. Chem. 10 (4): 553-7 (1999); and Zimmerman et al., Nucl. Med. Biol. 26 (8): 943-50 (1999). These are hereby incorporated by reference in their entirety.

  As noted above, the albumin fusion proteins of the invention can be modified either by natural processes (eg, post-translational processing) or chemical modification techniques well known in the art. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. The polypeptides of the invention can be, for example, branched as a result of ubiquitination, and the polypeptides can be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides can result from natural post-translational processes or can be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, flavin covalent bond, heme moiety covalent bond, nucleotide or nucleotide derivative covalent bond, lipid or lipid derivative covalent bond, phosphatidylinositol Covalent bond, crosslinking, cyclization, disulfide bond formation, demethylation, covalent bridge formation, cysteine formation, pyroglutamic acid formation, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, Iodination, methylation, mytilylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to proteins (eg, Giniru reduction), and ubiquitination. (For example, PROTEINS-STRUCTURE AND MOLECULAR PROPERIES, 2nd edition, TE Creighton, WH Freeman and Company, New York (1993); POST-TRANSTRATIONAL COVALENT. Press, New York, pp. 1-12 (1983); Seeifter et al., Meth Enzymol 182: 626-646 (1990); Rattan et al., Ann NY Acad Sci 663: 48-62 (1992)).

  Antibodies that bind to the albumin fusion proteins of the invention, and therapeutic proteins or fragments or variants thereof can be fused to a marker sequence (eg, a peptide to facilitate purification). In a preferred embodiment, the marker amino acid sequence is a hexahistidine peptide (eg, a tag provided, inter alia, in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91111), and Many are commercially available. Gentz et al., Proc. Natl. Acad. Sci. USA 86: 821-824 (1989), for example, hexahistidine provides convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, “HA” tags and “flag” tags. This “HA” tag corresponds to an epitope derived from influenza hemagglutinin protein (Wilson et al., Cell 37: 767 (1984)).

  Furthermore, the albumin fusion protein of the present invention can be used in therapeutic moieties (eg, cytotoxins (eg, cytostatic or cell killing factors)), therapeutic agents, or radioactive metal ions (eg, α-emitters (eg, 213Bi)). Can be combined. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin dioxy, Santron, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs thereof. Therapeutic agents include antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine, alkylating agents (eg, mechlorethamine, thioepa chlorambucil, Melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum (II) (DDP) cisplatin, anthracyclines (eg, , Daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (eg dactinomycin) Emissions (formerly actinomycin), bleomycin, mithramycin, and anthramycin, (Anthramycin) (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine) include, but are not limited to.

The conjugates of the invention can be used to modify a given biological response, and the therapeutic agent or drug component should not be construed as limited to classical chemotherapeutic agents. For example, the drug component can be a protein or polypeptide that possesses the desired biological activity.
Such proteins include, for example, toxins such as abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin; tumor necrosis factor, alpha interferon, beta interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, Apoptotic agents such as TNFα, TNFβ, AIM I (see WO 97/33899), AIM II (see WO 97/34911), Fas ligand (Takahashi et al., Int. Immunol., 6: 1567-1574 (1994)), VEGI (see International Publication No. WO 99/23105), thrombotic factors or anti-angiogenic factors, eg proteins such as angiostatin or endostatin Or; for example, lymphokines, interleukin 1 (“IL-1”), interleukin 2 (“IL-2”), interleukin 6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM”) -CSF "), granulocyte colony stimulating factor (" G-CSF "), or other growth factors such as growth factors. Techniques for conjugating such therapeutic components to proteins (eg, albumin fusion proteins) are well known in the art.

  Albumin fusion proteins can also be attached to a solid support, which is particularly useful for immunoassays or purification of polypeptides that are bound to, bound to, or associated with the albumin fusion proteins of the present invention. . Such solid supports include but are not limited to glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

  The albumin fusion protein can be used as a therapeutic agent, alone or in combination with cytotoxic factor (s) and / or cytokine (s), with or without the therapeutic component conjugated thereto.

  In an embodiment in which the albumin fusion protein of the present invention includes only the VH domain of an antibody that binds a therapeutic protein, the fusion protein with the VL domain of the same antibody that binds the therapeutic protein, the VH albumin fusion protein and the VL protein are post-translational , May be necessary and / or desirable to be co-expressed to associate (either covalently or non-covalently).

  In an embodiment in which the albumin fusion protein of the present invention includes only the VL domain of an antibody that binds a therapeutic protein, the VL albumin fusion protein and the VH protein are translated into the fusion protein with the VH domain of the same antibody that binds the therapeutic protein. Later, it may be necessary and / or desirable to co-express to associate (either covalently or non-covalently).

  Some therapeutic antibodies are bispecific, meaning that the antibody that binds the therapeutic protein is an artificial hybrid antibody having two different heavy / light chain pairs and two different binding sites. In order to create an albumin fusion protein corresponding to the therapeutic protein, it is possible to create an albumin fusion protein having an scFv fragment fused to both the N-terminus and the C-terminus of this albumin protein component. More specifically, the scFv fused to the N-terminus of albumin may correspond to one of the heavy chain / light chain (VH / VL) pairs of the original antibody that binds the therapeutic protein and is fused to the C-terminus of albumin The scFv produced can correspond to the other of the heavy / light chain (VH / VL) pairs of the original antibody that binds the therapeutic protein.

  Also provided by the present invention is a chemical modification of the albumin fusion protein of the present invention that may provide additional benefits such as increased solubility, stability and circulation time of the polypeptide, or reduced immunogenicity. Derivatives (see US Pat. No. 4,179,337). The chemical component for derivatization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. Albumin fusion proteins can be modified at random positions within the molecule, or at predetermined positions within the molecule, and can include one, two, three or more added chemical moieties.

  The polymer can be of any molecular weight and can be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa for ease of handling and manufacturing (the term “about” refers to several molecules in the preparation of polyethylene glycol. Indicating a weight that is somewhat greater or less than the given molecular weight). Other sizes are also desired for the desired therapeutic profile (eg, desired duration of sustained release, effects on biological activity, if present, ease of handling, degree or lack of antigenicity, and polyethylene glycol for therapeutic proteins or analogs) Other known effects). For example, the polyethylene glycol is about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10, 000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 45, 000, 50,000, 55,000, 60,000, 65,000, 70,00 It may have an average molecular weight of 75,000,80,000,85,000,90,000,95,000 or 100,000KDa,.

  As mentioned above, the polyethylene glycol can have a branched structure. Branched polyethylene is described, for example, in US Pat. No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56: 59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 18: 2745-2750 (1999); and Caliceti et al., Bioconjug. Chem. 10: 638-646 (1999), the disclosures of each of which are incorporated herein by reference.

  This polyethylene glycol molecule (or other chemical component) should be attached to the protein in view of its effect on the functional or antigenic domain of the protein. There are many attachment methods available to those skilled in the art, such as the method disclosed in EP 0 401 384, incorporated herein by reference (PEG attachment to G-CSF); Report PEGylation of GM-CSF using chloride, Malik et al., Exp. Hematol. 20: 1028-1035 (1992). For example, polyethylene glycol can be covalently linked through an amino acid residue via a reactive group, such as a free amino group or a carboxyl group. A reactive group is a group to which an activated polyethylene glycol molecule can bind. An amino acid residue having a free amino group can include a lysine residue and an N-terminal amino acid residue; an amino acid residue having a free carboxyl group can include an aspartic acid residue, a glutamic acid residue, and a C-terminal amino acid residue. May be included. Sulfhydryl groups can also be used as reactive groups for attaching polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.

  As suggested above, polyethylene glycol can be attached to the protein via a bond to any number of amino acid residues. For example, polyethylene glycol can be attached to the protein via a covalent bond to a lysine, histidine, aspartic acid, glutamic acid, or cysteine residue. One or more reactive chemicals may add polyethylene glycol, a specific amino acid residue of a protein (eg, lysine, histidine, aspartic acid, glutamic acid, or cysteine), or one or more types of amino acid residues of a protein (eg, Lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof).

  A protein that is chemically modified at the N-terminus may be specifically desired. As an illustration of the composition of the present invention, using polyethylene glycol, one skilled in the art will be able to determine the various polyethylene glycol molecules (by molecular weight, branching, etc.), the ratio of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mixture. The type of pegylation reaction to be performed, and the method of obtaining the selected N-terminal PEGylated protein may be selected. A method for obtaining an N-terminal PEGylated preparation (ie, separating this portion from other mono-PEGylated moieties if necessary) is by purifying the N-terminal PEGylated material from a population of PEGylated protein molecules. possible. Selective proteins chemically modified in N-terminal modification are reduced forms that utilize different reactivity of different types of primary amino groups (lysine to N-terminus) available for derivatization in specific proteins It can be achieved by alkylation. Under appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carboxyl group-containing polymer is achieved.

  As indicated above, pegylation of an albumin fusion protein of the invention can be accomplished by any number of means. For example, polyethylene glycol can be attached to the albumin fusion protein either directly or by an intervening linker. A linker-free system for attaching polyethylene glycol to proteins is described by Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9: 249-304 (1992); Francis et al., Intern. J. et al. of Hematol. 68: 1-18 (1998); U.S. Pat. No. 4,002,531; U.S. Pat. No. 5,349,052; WO 95/06058; and WO 98/32466 (the disclosures of each of which are incorporated herein by reference). Incorporated by reference).

One system for direct attachment of polyethylene glycol to amino acid residues of proteins without the use of an intervening linker is by modification of monomethoxypolyethylene glycol (MPEG) with tresyl chloride (ClSO ₂ CH ₂ CF ₃ ). The generated, tresylated MPEG is used. Upon reaction of the protein with tresylated MPEG, polyethylene glycol is directly attached to the amine group of the protein. Accordingly, the present invention includes a protein-polyethylene glycol conjugate produced by reacting a protein of the present invention with a polyethylene glycol molecule having a 2,2,2-trifluoroethanesulfonyl group. .

  Polyethylene glycol can also be attached to proteins using many different interventional linkers. For example, US Pat. No. 5,612,460 (the entire disclosures of which are incorporated herein by reference) discloses urethane linkers for linking polyethylene glycol to proteins. The protein-polyethylene glycol conjugate, where the polyethylene glycol is attached to the protein by a linker, was also activated with the protein and MPEG-succinimidyl succinate, 1,1′-carbonyldiimidazole. It can be produced by reaction with compounds such as MPEG, MPEG-2,4,5-trichloropenyl carbonate, MPEG-p-nitrophenol carbonate, and various MPEG-succinate derivatives. Many additional polyethylene glycol derivatives and reaction chemistries for conjugating polyethylene glycol to proteins are described in International Publication No. WO 98/32466, the entire disclosures of which are hereby incorporated by reference. PEGylated protein products produced using the reaction chemistry described herein are included within the scope of the present invention.

  The number of polyethylene glycol moieties attached to each albumin fusion protein of the invention (ie, the degree of substitution) can also vary. For example, the PEGylated protein of the present invention, on average, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, It can be linked to 17, 20 or more polyethylene glycol molecules. Similarly, the average degree of substitution is 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9 per protein molecule, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, Or a range of polyethylene glycol moieties, such as 18-20. Methods for determining the degree of substitution are described, for example, in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9: 249-304 (1992).

  The polypeptides of the present invention can be prepared by ammonium sulfate or ethanol precipitation, acid extraction, anion exchange or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography. Can be recovered and purified from chemical synthesis and recombinant cell culture by standard methods including, but not limited to, graphics. Most preferably, high performance liquid chromatography (“HPLC”) is used for purification. Well-known techniques for protein refolding can be used to regenerate the active conformation when the polypeptide is denatured during isolation and / or purification.

  The presence or amount of an albumin fusion protein of the invention can be determined using ELISA, an immunoassay well known in the art. In one ELISA, a useful protocol for detecting / quantifying the albumin fusion protein of the present invention is the step of coating an ELISA plate with an anti-human serum albumin antibody, blocking the plate to prevent non-specific binding. A step of washing the ELISA plate, adding a solution containing the albumin fusion protein of the present invention (at one or more different concentrations), a secondary anti-therapeutic protein-specific antibody linked to a detectable label Adding (as described herein or otherwise known in the art) and detecting the presence of secondary antibodies. In an alternative version of this protocol, the ELISA plate can be coated with an anti-therapeutic protein specific antibody and the labeled secondary reagent can be an anti-human albumin specific antibody.

(Use of polynucleotides)
Each of the polynucleotides identified herein can be used as a reagent in many ways. The following description should be considered exemplary and utilizes known techniques.

  The polynucleotide of the present invention is useful for producing the albumin fusion protein of the present invention. As described in more detail below, a polynucleotide of the invention (encoding an albumin fusion protein) is a cell, cell line that expresses an albumin fusion protein encoded by a polynucleotide encoding an albumin fusion protein of the invention. Or can be used in recombinant DNA methods useful in genetic engineering to create tissues.

  The polynucleotides of the invention are also useful in gene therapy. One purpose of gene therapy is to insert a normal gene into an organism with a defective gene in an attempt to correct the genetic defect. The polynucleotides disclosed in the present invention provide a means of targeting such genetic defects in a very accurate manner. Another purpose is to insert a new gene that was not present in the host genome, thereby creating a new trait in the host cell. Further non-exemplary examples of gene therapy methods encompassed by the present invention are described more closely elsewhere herein (eg, the section labeled “Gene Therapy” and Examples 63 and 64). See

(Use of polypeptides)
Each polypeptide identified herein can be used in a number of ways. The following description should be considered exemplary and utilizes known techniques.

  The albumin fusion proteins of the present invention can be expressed in tissue (eg, immunohistochemical assays such as ABC immunoperoxidase (Hsu et al., J. Histochem. Cytochem. 29: 577-580 (1981)) or cell types (eg, immunocytochemistry). Useful for providing immunological probes for differential identification of assays).

Albumin fusion proteins can be used to assay the level of polypeptide in a biological sample using classical immunohistological methods known to those skilled in the art (see, eg, Jalkanen et al., J. Cell. Biol. 101: 976-985 (1985); see Jalkanen et al., J. Cell. Biol. 105: 3087-3096 (1987)). Other methods useful for detecting gene expression of proteins include immunoassays (eg, enzyme linked immunosorbent assay (ELISA) and radioimmunoassay (RIA)). Suitable assay labels are known in the art and are enzyme labels (eg glucose oxidase); radioisotopes (eg iodine ( ¹³¹ I, ¹²⁵ I, ¹²³ I, ¹²¹ I), carbon ( ¹⁴ C). , Sulfur ( ³⁵ S), tritium ( ³ H), indium ( ^{115 m} In, ^{113 m} In, ¹¹² In, ¹¹¹ In), and technetium ( ⁹⁹ Tc, ^{99 m} Tc), thallium ( ²⁰¹ Ti), gallium ( ⁶⁸ Ga, ⁶⁷ Ga), palladium ( ¹⁰³ Pd), molybdenum ( ⁹⁹ Mo), xenon ( ¹³³ Xe), fluorine ( ¹⁸ F), ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁵⁹ Gd, ¹⁴⁹ Pm, ¹⁴⁰ La, ¹⁷⁵ Yb, ¹⁶⁶ Ho, ⁹⁰ Y ^{, 47 Sc, 186 Re, 188} Re, 142 Pr, 105 ^h, 97 Ru; luminescent labels (e.g., luminol); and fluorescent labels (e.g., fluorescein and rhodamine) include and biotin.

  The albumin fusion proteins of the present invention can also be detected in vivo by imaging. Antibody labels or markers for in vivo imaging of proteins include those detectable by radiography, nuclear magnetic resonance (NMR), or electron spin resonance (ESR). Suitable labels for radiography include radioisotopes (eg, barium or cesium) that emit detectable radiation but are not clearly harmful to the subject. Suitable markers for NMR and ESR include markers with detectable characteristic spins (eg, deuterium) that label nutrients generated in cell lines expressing the albumin fusion proteins of the invention. Can be incorporated into the albumin fusion protein.

Radioisotopes (eg, ¹³¹ I, ¹¹² In, ^99m Tc, ( ¹³¹ I, ¹²⁵ I, ¹²³ I, ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ^{115 m In, 113m In, 112 In,} 111 In), and technetium ^{(99 Tc,} 99m ^Tc), thallium ⁽²⁰¹ Ti), gallium ^{(68 Ga,} 67 ^Ga), palladium ⁽¹⁰³ Pd), molybdenum ⁽⁹⁹ Mo), xenon ⁽¹³³ Xe), fluorine ^{(18 F, 153 Sm, 177} Lu, 159 Gd, 149 Pm, 140 La, 175 Yb, 166 Ho, 90 Y, 47 Sc, 186 Re, 188 Re, 142 Pr, 105 Rh, 97 Ru), radiopaque material, or detected by nuclear magnetic resonance Albumin fusion proteins, labeled with a suitable detectable imaging moiety such as an active material, can be used in mammals to be examined for immune system disorders (eg, parenterally, subcutaneously, or intraperitoneally). It is understood in the art that the size of the subject and the imaging system used will determine the amount of imaging moiety needed to produce a diagnostic image. For human subjects, the amount of radioactivity injected is typically in the range of about 5-20 millicuries of ^99m Tc.The labeled albumin fusion protein is then preferably one or more receptors, A body (eg, organ, cell, etc.) on which a ligand or substrate (corresponding to that of a therapeutic protein used to make an albumin fusion protein of the invention) is placed Accumulated in a location in the extracellular space or extracellular matrix), or where the albumin fusion protein comprises at least a fragment or variant of a therapeutic antibody, then the labeled albumin fusion protein is preferably a therapeutic antibody ( Position in the body (eg organ, cell, extracellular space or extracellular matrix) where the polypeptide / epitope corresponding to that bound by (used to make the albumin fusion protein of the invention) is located In vivo tumor imaging is described by SW Burchiel et al., “Immunopharmacologicals of Radiolabeled Antibodies and Ther Fragments” (Tumor Imaging, Chapter 13: The Rad). iochemical Detection of Cancer, S.M. W. Burchiel and B.M. A. Edited by Rhodes, Masson Publishing Inc. (1982)). The protocols described herein can be readily modified by those skilled in the art for use with the albumin fusion proteins of the present invention.

  In one embodiment, the invention provides an albumin fusion protein of the invention associated with a heterologous polypeptide or nucleic acid (eg, a polypeptide and / or antibody encoded by a polynucleotide encoding an albumin fusion protein of the invention). Methods are provided for specific delivery of an albumin fusion protein of the invention to a cell upon administration. In one example, the present invention provides a method for delivering a therapeutic protein into a targeted cell. In another example, the invention relates to single-stranded nucleic acid (eg, antisense or ribozyme) or double-stranded nucleic acid (eg, DNA that can be integrated into the genome of a cell or can be replicated episomally and transcribed. ) Is provided for delivery to targeted cells.

  In another embodiment, the present invention provides a method for specific destruction of cells (eg, destruction of tumor cells) by administering an albumin fusion protein of the invention associated with a toxin or cytotoxic prodrug. provide.

“Toxins” are endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or cells or cell surfaces that cause cell death under defined conditions Means one or more compounds that bind and activate any molecule or enzyme not normally present. Toxins that can be used in accordance with the methods of the present invention include, but are not limited to: radioisotopes known in the art, compounds that bind to intrinsic or derived endogenous cytotoxic effector systems. (Eg, an antibody (or a complement-containing portion thereof)), thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, Pokeweed antiviral protein, alpha sarcin and cholera toxin. A “toxin” can also be a cytostatic or destructive agent, a therapeutic agent or a radioactive metal ion (eg, an α-emitter (eg, ²¹³ Bi)) or other radioisotope (eg, ¹⁰³ Pd, ¹³³ Xe , ¹³¹ I, ⁶⁸ Ge, ⁵⁷ Co, ⁶⁵ Zn, ⁸⁵ Sr, ³² P, ³⁵ S, ⁹⁰ Y, ¹⁵³ Sm, ¹⁵³ Gd, ¹⁶⁹ Yb, ⁵¹ Cr, ⁵⁴ Mn, ⁷⁵ Se, ¹¹³ Sn, ⁹⁰ Yttrium, ¹¹⁷ Tin, ¹⁸⁶ rhenium, ¹⁶⁶ holmium, and ¹⁸⁸ rhenium); luminescent labels (eg, luminol); and fluorescent labels (eg, fluorescein and rhodamine), and biotin. In certain embodiments, the invention provides for the specific destruction of cells (eg, the disappearance of tumor cells) by administering a polypeptide of the invention or an antibody of the invention related to the radioisotope ⁹⁰ Y. Provide a way. In another specific embodiment, the present invention provides for the specific destruction of cells (eg, disappearance of tumor cells) by administering a polypeptide of the invention or an antibody of the invention related to the radioisotope ¹¹¹ In. Provide a way for. In a more specific embodiment, the invention provides for the specific destruction of cells (eg, the disappearance of tumor cells) by administering a polypeptide of the invention or an antibody of the invention associated with radioisotope ¹³¹ I. Providing a method for

  Techniques known in the art can be applied to label the polypeptides of the present invention. Such techniques include the use of bifunctional binders (eg, US Pat. Nos. 5,756,065; 5,714,631; 5,696,239; 5,652). No. 5,361; No. 5,505,931; No. 5,489,425; No. 5,435,990; No. 5,428,139; No. 5,342,604; Nos. 5,274,119; 4,994,560; and 5,808,003, the contents of each of which are incorporated herein by reference in their entirety) However, it is not limited to this.

  The albumin fusion proteins of the present invention are useful for diagnosis, treatment, interference and / or prognosis of various disorders in mammals, preferably humans. Such disorders include, but are not limited to, those described herein under the heading “Biological activity” in the following section.

  Accordingly, the present invention provides a method of diagnosing a disorder, comprising: (a) assaying the expression level of a particular polypeptide in an individual's cells or fluids using the albumin fusion protein of the present invention. And (b) comparing the assayed polypeptide expression level with the standard polypeptide expression level, thereby increasing or decreasing the assayed polypeptide expression level compared to the standard polypeptide expression level. Process shown. For cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition to the onset of the disease or the disease before the actual clinical symptoms become apparent A means for detecting can be provided. This more definitive type of diagnosis may allow health professionals to use preventive or aggressive treatments earlier, thereby preventing the development or further progression of cancer.

  In addition, diseases or conditions (eg, neurological disorders, immune system disorders, muscular disorders, reproductive disorders, gastrointestinal disorders, lung disorders, cardiovascular disorders, kidney disorders, proliferative disorders and / or cancerous properties using albumin fusion proteins of the present invention) Diseases and conditions) can be treated or prevented. For example, the patient can reverse the absence or decreased level of a polypeptide (eg, insulin), supplement the absence or decreased level of a different polypeptide (eg, hemoglobin S, SOD, catalase for hemoglobin B) DNA repair proteins), inhibiting polypeptide activity (eg, oncogenes or tumor suppressors), activating polypeptide activity (eg, by binding to receptors), membrane binding for free ligands Reducing the activity of membrane-bound receptors by competing with the receptors (eg, soluble TNF receptors used in reducing inflammation) or producing the desired response (eg, inhibiting vascular growth, proliferating cells or For the enhancement of immune response to tissues) There are, polypeptide can be administered in the present invention.

  In particular, albumin fusion proteins comprising at least a fragment or variant of a therapeutic antibody can also be used to treat a disease (as described above and elsewhere herein). For example, administration of an albumin fusion protein comprising at least a fragment or variant of a therapeutic antibody can bind the polypeptide and / or a polypeptide to which the therapeutic antibody used to make the albumin fusion protein specifically binds. And / or may reduce the overproduction of polypeptides to which the therapeutic antibody used to make the albumin fusion protein specifically binds. Similarly, administration of an albumin fusion protein comprising at least a fragment or variant of a therapeutic antibody allows the therapeutic antibody used to make the albumin fusion protein to bind to a polypeptide that is bound to a membrane (receptor). Polypeptides that specifically bind can be activated.

  At least, the albumin fusion protein of the present invention can be used as a molecular weight marker for SDS-PAGE gels or molecular sieve gel filtration columns using methods well known to those skilled in the art. As a method of assessing host cell transformation, the albumin fusion proteins of the present invention can also be used to raise antibodies, which can then be used to treat therapeutic proteins from recombinant cells, albumin proteins, and / Or measure protein expression of the albumin fusion protein of the invention. In addition, the albumin fusion proteins of the present invention can be used to test the following biological activities:

(Diagnostic assay)
The compounds of the present invention are useful for the diagnosis, treatment, prevention and / or prediction of various disorders in mammals (preferably humans). Such disorders include the corresponding columns of Table 1, as well as “immunoassay”, “blood related disorders”, “hyperproliferative disorders”, “kidney disorders”, “cardiovascular disorders”, “breathing disorders” "Angiogenic activity", "Disease at the cellular level", "Wound healing and epithelial cell proliferation", "Neurological activity and neurological disease", "Endocrine disorders", "Reproductive system disorders", "Infectious disorders", Examples include, but are not limited to, those described for each therapeutic protein herein under the section headed “re-onset” and / or “gastrointestinal disorders”.

  For a number of disorders, a substantially altered (increased or decreased) level of gene expression results in cells or fluids (eg, serum, plasma, urine, semen, synovial fluid or medulla obtained from an individual having such a disorder. Fluid) can be detected relative to a “standard” gene expression level, ie, the expression level in a tissue or body fluid from an individual without a disorder. Accordingly, the present invention provides a diagnostic method useful during diagnosis of a disorder, the method measuring and measuring the expression level of a gene encoding a polypeptide in a tissue, cell or body fluid from an individual. Comparing the gene expression level with a standard gene expression level, whereby an increase or decrease in gene expression level compared to the standard is indicative of the disorder. These diagnostic assays can be performed in vivo or in vitro, such as on a blood sample, biopsy tissue, or autopsy tissue.

  The present invention is also useful as a predictive indicator, whereby patients who show an increase or decrease in gene expression suffer bad clinical results.

  “Assaying the expression level of a gene encoding a polypeptide” means that the level of a particular polypeptide, or the mRNA level encoding a polypeptide of the invention, is directly (eg, Measuring or evaluating, qualitatively or quantitatively, by determination or evaluation of absolute protein levels or mRNA levels) or relative (eg, by comparison to polypeptide or mRNA levels of a second biological sample) Intended. Preferably, the polypeptide level or mRNA level in the first biological sample is measured or evaluated and compared to a standard polypeptide level or mRNA level, which was obtained from an individual without a disorder. Obtained from a second biological sample or determined by averaging levels from a population of individuals who do not have an immune system disorder. As will be appreciated in the art, once a standard polypeptide level or mRNA level is known, it can be used repeatedly as a standard for comparison.

  A “biological sample” is any biology obtained from an individual, cell line, tissue culture, or other source that contains a polypeptide (including a portion thereof) or mRNA of the invention. Intended to be a sample. As indicated, biological samples include body fluids (eg, serum, plasma, urine, synovial fluid, and cerebrospinal fluid) and tissue supplies that have been found to express full-length polypeptides or fragments or mRNA thereof. Sources are mentioned. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. If the biological sample contains mRNA, tissue biopsy is the preferred source.

  Total cellular RNA can be obtained using any suitable technique, such as the one-step guanidinium-thiocyanate-phenol-chloroform method described by Chomczynski and Sacchi (Anal. Biochem. 162: 156-159 (1987)). Can be isolated from a sample. The level of mRNA encoding the polypeptide is then assayed using any suitable method. These include Northern blot analysis, S1 nuclease mapping, polymerase chain reaction (PCR), reverse transcription combined with polymerase chain reaction (RT-PCR), and reverse transcription combined with ligase chain reaction (RT-LCR).

  The invention also includes diagnostic assays, such as quantitative and diagnostic assays, that detect levels of a polypeptide in biological samples (eg, cells and tissues), including determination of normal and abnormal levels of the polypeptide. About. Thus, for example, a diagnostic assay according to the present invention for detecting overexpression of a polypeptide that binds to, is bound by, or is associated with an albumin fusion protein compared to a normal control tissue sample Can be used to detect the presence of a tumor. Assay techniques that can be used to determine levels of a polypeptide that binds to, is bound by, or associated with an albumin fusion protein of the invention in a sample derived from a host are known to those of skill in the art. It is well known. Such assay methods include radioimmunoassays, competitive binding assays, Western blot analysis, and ELISA assays. Assaying polypeptide levels in a biological sample is possible using any method known in the art.

Assaying polypeptide levels in a biological sample is possible using techniques. For example, polypeptide expression in tissues is determined by classical immunohistological methods (Jalkanen et al., J. Cell. Biol. 101: 976-985 (1985); Jalkanen, M. et al., J. Cell. Biol. 105: 3087-3096 (1987)). Other methods useful for detecting polypeptide gene expression include immunoassays (eg, enzyme linked immunosorbent assay (ELISA) and radioimmunoassay (RIA)). Suitable antibody assay labels are known in the art, and enzyme labels (eg, glucose oxidase) and radioisotopes (eg, iodine ( ¹²⁵ I, ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S) , Tritium ( ³ H), indium ( ¹¹² In), and technetium ( ^99m Tc) and fluorescent labels (eg, fluorescein and rhodamine), and biotin.

  The tissue or cell type to be analyzed generally includes a tissue or cell type that is known or predicted to express the gene of interest (eg, cancer, etc.). Protein isolation methods used herein are described, for example, in Harlow and Lane (Harlow, E. and Lane, D., 1988 “Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, Cold Spring Harbor, York, which is incorporated herein by reference in its entirety. Isolated cells can be from cell cultures or patients. Analysis of cells harvested from the culture can be an essential step in the evaluation of cells that can be used as part of cell-based gene therapy techniques or to test the effects of compounds on gene expression.

  For example, an albumin fusion protein can be used to quantitatively or qualitatively detect the presence of a polypeptide that binds to, is bound by, or associated with an albumin fusion protein of the invention. . This can be achieved, for example, by immunofluorescence techniques using albumin fusion proteins that are fluorescently labeled with light microscopic detection, flow cytometric detection, or fluorescence colorimetric detection.

  In a preferred embodiment, at least a fragment or variant of an antibody that specifically binds at least a therapeutic protein as disclosed herein (disclosed in Table 1) or otherwise known in the art. Albumin fusion proteins can be used to quantitatively or qualitatively detect the presence of a gene product, or a conserved variant or peptide fragment thereof. This can be achieved, for example, by immunofluorescence techniques using fluorescently labeled antibodies in combination with light microscopy, flow cytometry, or fluorometric detection.

  The albumin fusion protein of the present invention may be immunofluorescent, immunoelectron microscope or non-specific for in situ detection of a polypeptide that binds to, is bound by, or associated with an albumin fusion protein. It can also be used histologically as an immunological assay. In situ detection can be accomplished by removing a histological specimen from the patient and applying to it a labeled antibody or polypeptide of the invention. The albumin fusion protein is preferably applied by covering the labeled albumin fusion protein on the biological sample. Through the use of such procedures, it is possible to determine not only the presence of a polypeptide that binds to, binds to, or is associated with an albumin fusion protein, but also its distribution in the tested tissue. It is. Using the present invention, those skilled in the art will readily understand that any of a wide variety of histological methods (eg, staining procedures) can be modified, for example, to achieve in situ detection.

  Immunoassays and non-immunoassays that detect polypeptides that bind to, are bound by, or associated with albumin fusion proteins are typically gene products or conserved variants or peptides thereof Samples (eg, biological fluids, tissue extracts, freshly collected cells, or lysates of cells incubated in cell culture) in the presence of detectably labeled antibodies capable of binding fragments. Incubating as well as detecting bound antibody by any of a number of techniques well known in the art.

  The biological sample can be contacted with a solid support or carrier (eg, nitrocellulose) or other solid support that can immobilize cells, cell particles or soluble proteins. The support can then be washed with a suitable buffer and subsequently treated with the detectable albumin fusion protein of the invention. The solid support can then be washed twice with buffer to remove unbound antibody or polypeptide. If necessary, the antibody is then labeled. The amount of immobilized label on the solid support can then be detected by conventional methods.

  A “solid support or carrier” binds a polypeptide (eg, a polypeptide that binds to, is bound by, or is associated with an albumin fusion protein or an albumin fusion protein of the invention). Any support that is possible is contemplated. Well known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, gabbro, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material can have virtually any possible structural configuration so long as the bound molecule can bind to the polypeptide. Thus, the structure of the support can be spherical in the case of beads or cylindrical in the case of the inner surface of a test tube or the outer surface of a rod. Alternatively, the surface can be flat (eg, a sheet, a test strip, etc.). A preferred support comprises polystyrene beads. One skilled in the art can ascertain whether many other suitable carriers capable of binding an antibody or antigen are known or are identical by use of routine experimentation.

  The binding activity of a given lot of albumin fusion protein can be determined according to well-known methods. One skilled in the art can determine effective and optimal assay conditions for each determination by using routine experimentation.

  In addition to assaying polypeptide levels in a biological sample obtained from an individual, the polypeptide can also be detected in vivo by imaging. For example, in one embodiment of the invention, the albumin fusion protein of the invention is used to image diseased or neoplastic cells.

  The antibody label or marker for imaging the albumin fusion protein of the present invention in vivo includes those detectable by X-ray photography, NMR, MRI, CAT scan or ESR. For radiography, suitable labels include radioisotopes (eg, barium or cesium) that emit detectable radiation but are not clearly harmful to the subject. Suitable markers for NMR and ESR include markers with a detectable characteristic spin (eg, deuterium) that can be added to albumin fusion proteins by labeling nutrients for related hybridomas. Can be incorporated.

  In addition, albumin fusion proteins of the invention whose presence can be detected can be administered. For example, an albumin fusion protein of the invention labeled with a radio-opaque compound or other suitable compound can be administered and imaged in vivo as described above for labeled antibodies. Furthermore, such polypeptides can be used for in vitro diagnostic procedures.

Polypeptide specific labeled with an appropriate detectable imaging moiety (eg, radioisotope (eg, ¹³¹ I, ¹¹² In, ^99m Tc), radiopaque material, or material detectable by nuclear magnetic resonance) An antibody or antibody fragment is introduced (eg, parenterally, subcutaneously, or intraperitoneally) into a mammal to be tested for the disorder. It will be understood in the art that the size of the subject and the imaging system used will determine the amount of imaging moiety needed to produce a diagnostic image. In the case of a radioisotope moiety, for a human subject, the amount of radioactivity injected typically ranges from about 5 to 20 millicuries of ^99m Tc. The labeled albumin fusion protein is then subjected to a polypeptide or other substrate that binds to, is associated with, or is associated with the albumin fusion protein of the invention or the albumin fusion protein of the invention. Accumulate preferentially at locations in the body that contain it. In vivo tumor imaging is W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragment,” 198 (Tumor Imaging: The Radio Detection of Cancer. .

  One way in which the albumin fusion proteins of the invention can be detectably labeled is by similarly linking to a reporter enzyme and using this linked product in an enzyme immunoassay (EIA) (Voller, A. “The. Enzyme Linked Immunosorbent Assay (ELISA) "1978, Diagnostic Horizons 2: 1-7, Microbiological Associates, Quarterly Publications, Walkersville, MD, V); Clin. Pathol. 31: 507-520 (1978); Butler, J. et al. E. Meth. Enzymol. 73: 482-523 (1981); Maggio, E .; (Eds.) 1980, Enzyme Immunoassay, PTPaseC Press, Boca Raton, FL; Ishikawa, E .; (Eds.) 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo). A reporter enzyme that binds to the antibody reacts with an appropriate substrate (preferably a chromogenic substrate) in such a way as to generate a chemical moiety that can be detected, for example, by spectrophotometry, fluorescence or visible means. Reporter enzymes used to detectably label antibodies include malate dehydrogenase, staphylococcal nuclease, δ-5-steroid isomerase, yeast alcohol dehydrogenase, α-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish Examples include, but are not limited to, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Furthermore, this detection can be achieved by a colorimetric method using a chromogenic substrate for the reporter enzyme. Detection can also be accomplished by visual comparison of the extent of enzymatic reaction of the substrate compared to a similarly prepared standard.

Albumin fusion proteins can also be radiolabeled and used in any of a variety of other immunoassays. For example, it is possible to use albumin fusion proteins in radioimmunoassay (RIA) by radiolabeling albumin fusion proteins (eg, Weintraub, B., Principles of Radioimmunoassays, Seventeen Training Coins on RadioligandTandigandigan See Endocrine Society, March, 1986, which is incorporated herein by reference). The radioactive isotope can be detected by means including, but not limited to, a γ counter, a scintillation counter, or an autoradiography.
In addition, chelator molecules known in the art can be used to label albumin fusion proteins. The chelating agent can bind to the albumin fusion protein of the present invention and facilitate the labeling of the protein with a metal ion including a radionuclide or a fluorescent label.
For example, Subramanian, R.A. And Meares, C.I. F. , “Bifunctional Chelating Agents for Radiometal-labeled Monoclonal Antibodies,” (Cancer Imaging with Radiolabeled Antibodies, D. M. Goldenbel). , "Targeted delivery of radiolabeled imaging and therapeutic agents: bifunctional radiopharmaceuticals." Crit. Rev. Ther. Drug Carrier Syst. 16: 209-244 (1999); Srivastava S .; C. And Mease R. C. , "Progress in research on ligands, nucleotides and techniques for labeling monoclonal antibodies." Int. J. et al. Rad. Appl. Instrum. B 18: 589-603 (1991); and Liu, S .; And Edwards, D .; S. , “Bifunctional chelators for therapeutic Lanthanide radiopharmaceuticals.” Bioconjug. Chem. 12: 7-34 (2001). Any chelating agent that can be covalently linked to the albumin fusion protein can be used in accordance with the present invention. The chelating agent can further comprise a linker moiety that binds to the chelating moiety for the albumin fusion protein.

In one embodiment, the albumin fusion protein of the invention comprises an acyclic (chelating agent (eg, diethylenetriamine-N, N, N ′, N ″, N ″ -pentaacetic acid (DPTA), an analog of DPTA, And a derivative of DPTA, as a non-limiting example,
Chelating agents include 2- (p-isothiocyanate benzyl) -6-methyldiethylenetriaminepentaacetic acid (1B4M-DPTA, also known as MX-DTPA), 2-methyl-6- (ρ-nitrobenzyl) -1,4, 7-triazaheptane-N, N, N ′, N ″, N ″ -pentaacetic acid (nitro-1B4M-DPTA or nitro-MX-DTPA);
(P-isothiocyanate benzyl) -cyclohexyldiethylenetriaminepentaacetic acid (CHX-DTPA) or N- [2-amino-3- (ρ-nitrophenyl) propyl] -trans-cyclohexane-1,2-diamine-N, N It can be ', N ″ -pentaacetic acid (nitro-CHX-A-DTPA).

In another embodiment, an albumin fusion protein of the invention comprises an acyclic terpyridine chelator (eg, 6,6 ″ -bis [[N, N ′, N ″, N ″ -tetra (carboxymethyl ) Amino] methyl] -4 '-(3-amino-4-methoxyphenyl) 2,2':
6 ', 2 "-terpyrzine (TMT-amine)).

In a specific embodiment, the macrocyclic chelator that binds to the albumin fusion protein of the invention is 1,4,7,10-tetraazacyclododecane-N, N ′, N ″, N ′ ″-tetraacetic acid. (DOTA). In another specific embodiment, examples of linker molecules useful for DOTA binding to a polypeptide that binds to an albumin fusion protein of the invention via a linker molecule are generally known in the art—for example DeNardo et al., Clin. Cancer Res. 4 (10): 2483-90, 1998; Peterson et al., Bioconjug. Chem. 10 (4): 553-7, 1999; and Zimmerman et al., Nucl. Med. Biol. 26 (8): 943-50 (the entirety of which is incorporated herein by reference). In addition, US Pat. Nos. 5,652,361 and 5,756,065, which disclose chelating agents that can bind to antibodies and methods for making and using them, are hereby incorporated by reference in their entirety. Incorporated by reference. Through U.S. Pat. Nos. 5,652,361 and 5,756,065, which focus on the conjugation of chelators to antibodies, one skilled in the art can easily adapt the methods disclosed therein and A chelating agent can be attached to the polypeptide.

Bifunctional chelators based on macrocyclic ligands where the linkage is attached to the carbon skeleton of the ligand via an activation arm, or functional group, can be utilized as described by: Moi et al. Amer. Chem. Soc. 49: 2639 (1989) (2-p-nitrobenzyl-1,4,7,10-tetraazacyclododecane-N, N ′, N ″, N ′ ″-tetraacetic acid); V. Deshpande et al. Nucl. Med. 31
: 473 (1990); Ruser et al., Bioconj. Chem. 1: 345 (1990); J. et al. Broan et al. C. S. Chem. Comm. 23: 1739 (1990); J. et al. Anderson et al. Nucl. Med. 36: 850 (1995).

  In one embodiment, a macrocyclic chelator (eg, a polyaza macrocyclic chelator) (optionally comprising one or more carboxyl groups, amino groups, hydroxamate groups, phosphanate groups, or phosphate groups) Binds to the albumin fusion protein of the present invention. In another embodiment, the chelator is a chelator selected from the group consisting of DOTA, an analog of DOTA, and a derivative of DOTA.

  In one embodiment, suitable chelator molecules that can bind to the albumin fusion proteins of the invention include DOXA (1-oxa-4,7,10-triazacyclododecane triacetic acid), NOTA (1, 4, 7-triazanonanetriacetic acid), TETA (1,4,8,11-tetraazacyclotetradecanetetraacetic acid), and THT (4 ′-(3-amino-4-methoxy-phenyl) -6,6 ″ -Bis (N ′, N′-dicarboxymethyl-N-methylhydrazino) -2,2 ′: 6 ′, 2 ″ -terpyridine), and analogs and derivatives thereof. For example, Ohmono et al. Med. Chem. 35: 157-162 (1992); Kung et al., J. Biol. Nucl. Med. 25: 326-332 (1984); Jurison et al., Chem. Rev. 93: 1137-1156 (1993); and US Pat. No. 5,367,080. Other suitable chelating agents include U.S. Pat. Nos. 4,647,447; 4,687,659; 4,885,363; EP-A-71564; WO89 / 00557; and EP- A chelating agent disclosed in A-232751 may be mentioned.

  In another embodiment, suitable macrocyclic carboxylic acid chelating agents that can be used in the present invention include 1,4,7,10-tetraazacyclododecane-N, N ′, N ″, N ′ ″ — Tetraacetic acid (DOTA); 1,4,8,12-tetraazacyclopentadecane-N, N ′, N ″, N ′ ″-tetraacetic acid (15N4); 1,4,7-tetraazacyclononane- N, N ′, N ″ -triacetic acid (9N3); 1,5,9-triazacyclododecane-N, N ′, N ″ -triacetic acid (12N3); and 6-bromoacetamido-benzyl-1 4,8,11-tetraazacyclotetradecane-N, N ′, N ″, N ′ ″-tetraacetic acid (BAT).

  A preferred chelating agent capable of binding to the albumin fusion protein of the present invention is α- (5-isothiocyanato-2-methoxyphenyl) -1,4,7,10-tetraazacyclododecane-1,4,7,10. Tetraacetic acid (also known as MeO-DOTA-NCS). A salt or ester of α- (5-isothiocyanato-2-methoxyphenyl) -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid may also be used.

The albumin fusion proteins of the present invention that are covalently bound to a chelator as described are (through the coordination site of the chelator) using a radionuclide suitable for therapeutic purposes, diagnostic purposes, or therapeutic and diagnostic purposes. Can be labeled. Examples of suitable metals include Ag, At, Au, Bi, Cu, Ga, Ho, In, Lu, Pb, Pd, Pm, Pr, Rb, Re, Rh, Sc, Sr, Tc, Tl, Y, And Yb. Examples of radionuclides used for diagnostic purposes are Fe, Gd, ¹¹¹ In, ⁶⁷ Ga, or ⁶⁸ Ga. In another embodiment, the radionuclide used for diagnostic purposes is ¹¹¹ In or ⁶⁷ Ga. Examples of radionuclides used for therapeutic purposes are ¹⁶⁶ Ho, ¹⁶⁵ Dy, ⁹⁰ Y, ^{115 m} In, ⁵² Fe, or ⁷² Ga. In one embodiment, the radionuclide used for therapeutic purposes is ¹⁶⁶ Ho or ⁹⁰ Y. Examples of radionuclides used for both therapeutic and diagnostic purposes include ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁵⁹ Gd, ¹⁷⁵ Yb, or ⁴⁷ Sc. In one embodiment, the radionuclide is ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁷⁵ Yb, or ¹⁵⁹ Gd.

Preferred metal radionuclides include ⁹⁰ Y, ^99m Tc, ¹¹¹ In, ⁴⁷ Sc, ⁶⁷ Ga, ⁵¹ Cr, ¹⁷⁷ mSn, ⁶⁷ Cu, ¹⁶⁷ Tm, ⁹⁷ Ru, ¹⁸⁸ Re, ¹⁷⁷ Lu, ¹⁹⁹ Au, ⁴⁷ Sc, ⁶⁷ Ga, ⁵¹ Cr, ¹⁷⁷ mSn, ⁶⁷ Cu, ¹⁶⁷ Tm, ⁹⁵ Ru, ¹⁸⁸ Re, ¹⁷⁷ Lu, ¹⁹⁹ Au, ²⁰³ Pb and ¹⁴¹ Ce.

In certain embodiments, an albumin fusion protein of the invention to which a chelating agent is covalently bonded using a metal ion selected from the group consisting of ⁹⁰ Y, ¹¹¹ In, ¹⁷⁷ Lu, ¹⁶⁶ Ho, ²¹⁵ Bi, and ²²⁵ Ac. Can be labeled.

In addition, gamma-emitting radionuclides (eg, ^99m Tc, ¹¹¹ In, ⁶⁷ Ga, and ¹⁶⁹ Yb) are improved under research for diagnostic imaging, but beta emitters (eg, ⁶⁷ Cu, ¹¹¹ Ag, ¹⁸⁶ Re, and ⁹⁰ Y) are used for applications in tumor therapy.
Other useful radionuclides include gamma emitters such as ^99m Tc, ¹¹¹ In, ⁶⁷ Ga, ¹⁶⁹ Yb and beta emitters (eg ⁶⁷ Cu, ¹¹¹ Ag, ¹⁸⁶ Re, ¹⁸⁸ Re and ⁹⁰ Y) and purposes Other radionuclides (e.g. ²¹¹ At, ²¹² Bi, ¹⁷⁷ Lu, ⁸⁶ Rb, ¹⁰⁵ Rh, ¹⁵³ Sm, ¹⁹⁸ Au, ¹⁴⁹ Pm, ⁸⁵ Sr, ¹⁴² Pr, ²¹⁴ Pb, ¹⁰⁹ Pd, ¹⁶⁶ Ho, ²⁰⁸ Tl, And ⁴⁴ Sc). The albumin fusion protein of the present invention covalently bound to a chelating agent can be labeled using the radionuclide described above.

  In another embodiment, an albumin fusion protein of the invention covalently linked to a chelator is a paramagnetic metal ion (eg, atomic numbers 21-29, 42, 43, 44, or 57) including transition metal and lanthanide metal ions. Metals having ~ 71, in particular Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and (Lu ions). Paramagnetic metals used in compositions for magnetic response imaging include elements having atomic numbers 21-29, 42, 44 and 58-70.

  In another embodiment, the albumin fusion protein of the present invention covalently attached to a chelator comprises a fluorescent metal ion (lanthanide, particularly La, Ce, Pr, Nd, Pm, Sm, Eu (eg, 152 Eu), Gd, Tb, Including Dy, Ho, Er, Tm, Yb, and Lu).

  In another embodiment, an albumin fusion protein of the invention that covalently binds to a chelator can be labeled with a reporter (which can include Mo, Bi, Si, and W atoms) containing heavy metals.

  It is also possible to label the albumin fusion protein with a fluorescent compound. If the fluorescently labeled albumin fusion protein is exposed to light of the appropriate wavelength, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde and fluorescamine.

Albumin fusion proteins can also be detectably labeled with fluorescent metals such as ¹⁵² Eu or other lanthanide series. These metals can be bound to albumin fusion proteins using metal chelates such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (ETDA).

  Albumin fusion proteins can also be detectably labeled by binding to a chemiluminescent compound. The presence of the chemiluminescent tagged albumin fusion protein is then determined by detecting the presence of luminescence that occurs during the course of the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, thermetic acridinium ester, imidazole, acridinium salt and oxalate ester.

Similarly, bioluminescent compounds can be used to label the albumin fusion proteins of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems where catalytic proteins increase the efficiency of chemiluminescent reactions. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for labeling purposes are luciferin, luciferase and aequorin.
(Transgenic organism)
Also included in the invention are transgenic organisms that express the albumin fusion proteins of the invention. Transgenic organisms are generally organisms that have been modified into recombinant, exogenous or transferred cloned genetic material. Such genetic material is often referred to as a transformant. The nucleic acid sequence of the transformant can include one or more transcription control sequences and other nucleic acid sequences (eg, introns) that may be required for optimal expression and secretion of the encoded protein. Transformants express the expression of an encoded protein in a manner that facilitates regeneration from the organism or product produced by the organism (eg, organism's milk, blood, urine, egg, hair or seed). Can be designed to dominate. A transformant (can consist of a nucleic acid sequence derived from the same or a heterologous genome as the target animal species. The transformant can be at a genomic location where a particular nucleic acid sequence is not normally found otherwise. Or at the normal position of the transformant.

  The term “germline transgenic organism” refers to a transgenic organism into which genetic changes or genetic information has been introduced into the germline, thereby transferring the genetic information to the offspring. Give the ability. If such offspring actually have some or all of such changes or genetic information, they are also transgenic organisms. The change or genetic information may be different from the species of the organism to which the recipient belongs, may differ only from the particular recipient individual, or may be genetic information already possessed by the recipient. In the last case, the altered or derived gene can be expressed differently than the native gene.

  The transgenic organism can be a transgenic animal or a transgenic plant. Transgenic animals can be made in a variety of different ways, including transfection, electroporation, microinjection, gene targeting in embryonic stem cells and recombinant viral infection and recombinant retroviral infection (US Pat. No. 4,736). No. 866; No. 5,602,307; Mullins et al. (1993) Hypertension 22 (4): 630-633; Brenin et al. (1997) Surg. Oncol. 6 (2) 99-110; Tuan (ed.), Recombinant See Gene Expression Protocols, Methods in Molecular Biology No. 62, Humana Press (1997)). The method of introducing a nucleic acid fragment into a recombinant competent mammalian cell can be any method advantageous for cotransformation of multiple nucleic acid molecules. Detailed procedures for producing transgenic animals are readily available to those of skill in the art and include disclosures in US Pat. Nos. 5,489,743 and 5,602,307.

  A number of recombinant or transgenic mice have been generated which are mice that express activated oncogene sequences (US Pat. No. 4,736,866); monkey SV40 T antigen sequence expressing mice (US Pat. 728,915); mice lacking expression of interferon regulatory factor 1 (IRF-1) (US Pat. No. 5,731,490); mice exhibiting dopaminergic dysfunction (US Pat. No. 5,723,719) Mice that express at least one human gene involved in blood pressure regulation (US Pat. No. 5,731,489); mice that show greater similarity to the conditions shown in naturally occurring Alzheimer's disease (US Pat. No. 5,720); 936); mice with reduced ability to mediate cell adhesion (US Pat. No. 5,602,307); Mice carrying the simulating growth hormone gene (Clutter et al. (1996) Genetics 143 (4): 1753-1760); or mice capable of generating a sufficient human antibody response (McCarthy (1997) The Lancet 349 (9049): 405).

  Mice and rats are animals selected for many transgenic studies, but in some instances it is preferred or even necessary to use alternative animal species. Transgenic procedures are successfully utilized in a variety of non-mouse animals, including sheep, goats, pigs, dogs, cats, monkeys, chimpanzees, hamsters, rabbits, cattle and guinea pigs (eg, Kim et al. (1997). Mol.Reprod.Dev.46 (4): 515-526; Houdebine (1995) Reprod.Nutr.Dev.35 (6): 609-617; Petters (1994) Reprod.Fertil.Dev.6 (5): 643 -645; Schnieke et al. (1997) Science 278 (5346): 2130-2133; and Amoah (1997) J. Animal Science 75 (2): 578-585)).

  In order to direct the secretion of the transformant-encoded protein of the present invention into the milk of transgenic mammals, the transformants can be placed under the control of a promoter that is preferentially activated in mammalian epithelial cells. . Promoters that regulate milk protein-encoding genes are preferably casein, β-lactoglobulin, whey acid protein, or lactalbumin promoters (eg, DiTullio (1992) BioTechnology 10: 74-77; Clark et al.). (1989) BioTechnology 7: 487-492; Gorton et al. (1987) BioTechnology 5: 1181-1187; and Soulier et al. (1992) FEBS Letts. 297: 13). The selected transgenic mammal produces large amounts of milk and has a long lactation period (eg, goat, cow, camel or sheep).

The albumin fusion proteins of the invention can also be expressed in transgenic plants (eg, plants in which a DNA transformant has been inserted into the nuclear or plastid genome).
Plant transformation procedures used to induce exogenous nucleic acids into plant cells or protoplasts are known in the art. See, generally, Methods in Enzymology Vol. 153 ("Recombinant DNA Part D") 1987, edited by Wu and Grossman, Academic Press and European Patent Application EP 695554. Methods for generating genetically engineered plants are further described in US Pat. Nos. 5,283,184, 5,482,852, and European Patent Application EP 693 554, all of which are hereby incorporated by reference. Incorporated by reference).

(Pharmaceutical composition or therapeutic composition)
The albumin fusion proteins of the invention or their formulations can be administered by any conventional method, including parenteral (eg, subcutaneous or intramuscular) or intravenous infusion. Treatment can consist of a single dose or multiple doses over a period of time.

  The albumin fusion proteins of the invention can be administered alone, but preferably are present as a pharmaceutical formulation with one or more acceptable carriers. This carrier should be “acceptable” in the sense of being consistent with albumin fusion proteins and should not be detrimental to their recipients. Typically, the carrier is sterilized and pyrogen free water or saline. Due to their extended shelf life in solution, the albumin fusion proteins of the present invention can be separated from aqueous carriers (eg, sterile pyrogen water, saline or solutions due to their extended shelf life in solution. Are particularly well suited for formulations in isotonic solutions). For example, a pharmaceutical composition of the invention can be well formulated in advance in aqueous form, for example, weeks or months or longer before being dispersed.

  For example, formulations containing albumin fusion proteins can be prepared in view of the extended shelf life of albumin fusion proteins in aqueous formulations. As described above, after fusion to HA, the shelf life of many of these therapeutic proteins is significantly increased or postponed.

  In examples where aerosol administration is appropriate, the albumin fusion proteins of the invention can be formulated as an aerosol using standard procedures. The term “aerosol” includes any gas-borne suspension phase of an albumin fusion protein of the invention that can be inhaled into the bronchiole or nasal route. In particular, the aerosol comprises a gas-bearing suspension of water droplets of the albumin fusion protein of the present invention, as produced in a metered dose inhaler or nebulizer, or a mist nebulizer. Aerosols also include dry powder compositions of the compounds of the invention suspended in air or other carrier gas that can be delivered by inhalation, for example, from an inhalation device. Ganderton and Jones, Drug Delivery to the Respiratory Tract, Elis Horwood (19-87); Toxicol. See Methods 27: 143-159.

  The formulations of the present invention may also typically be non-immunogenic due in part to the use of components of albumin fusion proteins derived from the appropriate species. For example, for human use, both the therapeutic protein and the albumin portion of the albumin fusion protein are typically human. In some cases where any component is of non-human origin, that component can be humanized by substitution of a major amino acid so that the specific epitope is rather exogenous to the human immune system. Become a natural human.

  The formulations may conveniently be present in unit dosage form and may be prepared by any method well known in the art of pharmacy. Such methods include the step of combining an albumin fusion protein with a carrier that constitutes one or more accessory ingredients. In general, a formulation is prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, forming the product.

Formulations suitable for parenteral administration include aqueous sterile infusion solutions or non-aqueous sterile infusion solutions that may include antioxidants, buffers, bacteriostats and solutions that make the formulation suitable for the intended recipient. And aqueous sterile or non-aqueous sterile infusion solutions that may contain suspending and thickening agents. The formulation can be in unit dose containers or multiple dose containers (eg, sealed ampoules, vials or syringes) and requires only the addition of a sterile carrier (eg, water for injection) just prior to use. And can be stored in a lyophilized (lyophilic) state.
Extemporaneous injection solutions and suspensions can be prepared from sterile powders. The dosage formulation is treated at a lower molar concentration or lower dosage compared to a non-fusion standard formulation for a therapeutic protein that provides the extended serum half-life exhibited by many albumin fusion proteins of the present invention. A portion of the protein can be included.

  By way of example, if the albumin fusion protein of the invention contains one of the proteins listed in the “Therapeutic protein: X” column of Table 1 as one or more therapeutic protein regions, compared to the half-life of native hGH. Thus, while considering extended serum half-life and albumin fusion protein half-life, the dosage form can be calculated based on the effectiveness of the albumin fusion protein proportional to the effectiveness of hGH. Growth hormone is typically administered at 0.3-30.0 IU / kg / week (e.g., 0.9-12.0 IU / kg / week) and is administered three times over one year or more. Or given in 7 divided doses. In albumin fusion proteins consisting of full-length HA fused to full-length GH, equal doses indicate a greater weight of the factor in terms of units, but the number of doses can be reduced (eg, twice a week, Once a week or less).

  The formulations or compositions of the invention can be packaged with instructions or a package insert that refers to the extended half-life of the albumin fusion protein component, or both can be included in a kit. For example, such instructions or packaging inserts note the recommended storage conditions (eg, time, temperature, and light), taking into account the prolonged or extended half-life of the albumin fusion protein of the invention. To turn. Such instructions or packaging inserts may also be used for certain advantages of the albumin fusion proteins of the invention, such as formulations that may be sought to be used in the field (outside regulated hospital, clinic or office conditions). Attention to ease of storage). As noted above, the formulations of the present invention can be in aqueous form and stored in an environment that is not without ideal loss of therapeutic activity but is never ideal.

  The albumin fusion proteins of the present invention can also be included as a nutritional supplement. For example, a particular albumin fusion protein of the invention can be administered in a natural product (milk or dairy product obtained from a transgenic mammal expressing the albumin fusion protein). Such compositions can also include a plant or plant product obtained from a transgenic plant that expresses an albumin fusion protein. Albumin fusion proteins can also be provided in powder or tablet form with or without other known additives, carriers, bulking agents and diluents. Nutritional support is available from Scott Hegenhart, Food Product Design, Dec. 1993.

  The present invention also includes administering to a subject an effective amount of an albumin fusion protein of the present invention or a polynucleotide encoding an albumin fusion protein of the present invention (albumin fusion polynucleotide) in a pharmaceutically acceptable carrier, Provided are methods for treating and / or preventing a disease or disorder (eg, any one or more diseases or disorders disclosed herein).

  Albumin fusion proteins and / or polynucleotides are known to the individual patient's clinical condition (especially the side effects of treatment with albumin fusion protein and / or polynucleotide alone), delivery site, administration method, dosing schedule and to those skilled in the art Take into account other factors and prescribe and administer in a manner that complies with good medical practice. Therefore, the target “effective amount” in the present specification is determined by taking such consideration into consideration.

  As a general suggestion, the total pharmaceutically effective amount of albumin fusion protein administered parenterally per dose is in the range of about 1 μg / kg / day to 10 mg / kg / day of patient body weight, This is left to therapeutic discretion. More preferably, for this hormone, this dose is at least 0.01 mg / kg / day, most preferably between about 0.01 mg / kg / day and about 1 mg / kg / day for humans. For continuous administration, typically the albumin fusion protein is injected 1 to 4 times daily at a dosage rate of about 1 μg / kg / hour to about 50 μg / kg / hour or continuous subcutaneous infusion (eg, using a minipump). ). Intravenous bag solutions may also be used. The duration of treatment necessary to observe the change and the post-treatment interval at which a response occurs appears to vary depending on the desired effect.

  Albumin fusion protein and / or polynucleotide can be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (such as by powder, ointment, gel, infusion, or transdermal patch) ), Administered orally or as an oral or nasal spray. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulant or any type of formulation adjuvant. The term “parenteral” as used herein refers to modes of administration including intravenous and intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injections and infusions.

  The albumin fusion proteins and / or polynucleotides of the present invention are also suitably administered by a sustained release system. Suitable examples of sustained release albumin fusion proteins and / or polynucleotides are oral, rectal, parenteral, intracisternal, intravaginal, intraperitoneal, topical (powder, ointment, gel, infusion, or transdermal (Via skin patch), administered by mouth, orally or as a nasal spray. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulant or any type of formulation adjuvant. As used herein, the term “parenteral” refers to modes of administration including intravenous and intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injections and infusions.

  Further examples of sustained release albumin fusion proteins and / or polynucleotides include suitable polymeric materials (eg, semi-permeable polymeric matrices (eg, films or microcapsules) in the form of molded articles), suitable hydrophobic materials (eg, For example, as an emulsion in an acceptable oil) or ion exchange resins, and low soluble derivatives (eg, low soluble salts).

  Sustained release matrices include polylactide (US Pat. No. 3,773,919, EP 58,481), a copolymer of L-glutamic acid and γ-ethyl-L-glutamate (Sidman et al., Biopolymers 22: 547-556 (1983)). ), Poly (2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater. Res. 15: 167-277 (1981), and Langer, Chem. Tech. 12: 98-105 (1982)), ethylene vinyl. Acetate (Langer et al., Ibid.) Or poly-D-(-)-3-hydroxybutyric acid (EP133,988).

  Sustained release albumin fusion proteins and / or polynucleotides also include albumin fusion proteins and / or polynucleotides of the invention encapsulated in liposomes (generally, Langer, Science 249: 1527-1533 (1990); Treat et al. , Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berstein and Fiddler (eds.), Liss, New York, pages 317-327 and 353-365 (1989)). Liposomes containing albumin fusion proteins and / or polynucleotides are prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA) 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. (USA) 77: 4030-4034 (1980); EP52,322; EP36,676; EP88,046; EP143,949; EP142,641; Japanese Patent Application No. 83-118008; US Patent No. 4,485,045 No. and No. 4,544,545; and EP No. 102,324. Usually, liposomes are small (about 200-800 cm) unilamellar type, where the lipid content is greater than about 30 mol% cholesterol and the selected proportion is adjusted for optimal therapeutic agents. .

  In still further embodiments, the albumin fusion proteins and / or polynucleotides of the invention are delivered by a pump (Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14: 201 (1987); Buchwald et al. Surgery 88: 507 (1980); Saudek et al., N. Engl. J. Med. 321: 574 (1989)).

  Other controlled release systems are discussed in the review by Langer (Science 249: 1527-1533 (1990)).

  For parenteral administration, in one embodiment, generally, the albumin fusion protein and / or polynucleotide is formulated in a desired degree of purity, in a pharmaceutically acceptable carrier, i.e., the dosage and concentration used. It is formulated by mixing in a unit dosage injectable form (solution, suspension or emulsion) with one that is not toxic to the entry and compatible with the other ingredients of the formulation. For example, the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to therapeutic agents.

  In general, a formulation is prepared by contacting albumin fusion protein and / or polynucleotide uniformly and intimately with a liquid carrier or a finely divided solid carrier or both. Next, if necessary, the product is shaped into the desired formulation. Preferably, the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Nonaqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as are liposomes.

  The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such substances are not toxic to the recipient at the dosages and concentrations used, such as phosphate, citrate, succinate, acetic acid and other organic acids or their salts. Buffering agents; antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides (eg, polyarginine or tripeptides); proteins such as serum albumin, gelatin or immunoglobulins; polyvinylpyrrolidone Hydrophilic polymers such as: amino acids such as glycine, glutamic acid, aspartic acid or arginine; monosaccharides, disaccharides and other carbohydrates including cellulose or derivatives thereof, glucose, mannose or dextrin; chelating agents such as EDTA Sugar sugars such as mannitol or sorbitol Lumpur; counterions such as sodium; and / or polysorbate include nonionic surfactants such as poloxamers or PEG.

  Albumin fusion proteins are typically formulated in such vehicles at a concentration of about 0.1 mg / ml to 100 mg / ml, preferably 1-10 mg / ml, at a pH of about 3-8. It will be appreciated that by using the specific excipients, carriers or stabilizers described above, a polypeptide salt is formed.

  Any pharmaceutical agent used for therapeutic administration can be sterile. Aseptic conditions are easily achieved by filtration through sterile filtration membranes (eg, 0.2 micron membranes). Generally, the albumin fusion protein and / or polynucleotide is placed in a container having a sterile access port, for example, an intravenous solution bag or vial with a stopper that can be punctured with a hypodermic needle.

  Albumin fusion proteins and / or polynucleotides are usually stored in unit dose or multi-dose containers, such as sealed ampoules or vials, as aqueous solutions or lyophilized formulations for reconstitution. As an example of a lyophilized formulation, a 10 ml vial is filled with 5 ml of sterile filtered 1% (w / v) albumin fusion protein and / or aqueous polynucleotide solution and the resulting mixture is lyophilized. Lyophilized albumin fusion protein and / or polynucleotide is reconstituted with bacteriostatic water for injection to prepare an infusion solution.

  In a specific and preferred embodiment, the albumin fusion protein formulation comprises 0.01 M sodium phosphate, 0.15 mM sodium chloride, 0.16 micromoles sodium octoate per mg of fusion protein, 15 micrograms / milliliter polysorbate 80, pH 7. 2 is included. In another specific and preferred embodiment, the albumin fusion protein formulation comprises 0.01 M sodium phosphate, 0.15 mM sodium chloride, 0.16 micromolar sodium octoate per mg of fusion protein, 15 micrograms / milliliter polysorbate 80, Consists of pH 7.2. The pH and buffer are selected to formulate to physiological conditions, and salt is added as a tonic. Sodium octanoate was selected due to its reported ability to increase the thermal stability of proteins in solution. Finally, polysorbate was added as a gene surfactant that lowers the surface tension of the solution and reduces non-specific adsorption of the albumin fusion protein to the container seal system.

  The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of an albumin fusion protein and / or polynucleotide of the invention. A notice of the form prescribed by a government agency that regulates the manufacture, use or sale of a medicinal product or biological product may be attached to such a container, and this notice may be attached to the government regarding the manufacture, use or sale for human administration. Represents institutional approval. In addition, albumin fusion proteins and / or polynucleotides can be used in combination with other therapeutic compounds.

  The albumin fusion proteins and / or polynucleotides of the invention can be administered alone or in combination with an adjuvant. Adjuvants that can be administered with the albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to: alum, alum + deoxycholic acid (ImmunoAg), MTP-PE (Biocine Corp.), Non-viable preparations of QS21 (Genentech, Inc.), BCG (eg THERACYS®), MPL and Corynebacterium parvum. In certain embodiments, the albumin fusion proteins and / or polynucleotides of the invention are administered in combination with alum. In another specific embodiment, the albumin fusion protein and / or polynucleotide of the invention is administered in combination with QS-21. Additional adjuvants that can be administered with the albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to: monophosphoryl lipid immunomodulators, AdjuVax 100a, QS-21, QS-18, CRL1005 , Aluminum salt, MF-59, and Virosomal adjuvant technology. Vaccines that can be administered with albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to: MMR (measles, mumps, rubella), polio, chickenpox Tetanus / diphtheria, hepatitis A, hepatitis B, influenza B, whooping cough, pneumonia, influenza, Lyme disease, rotavirus, cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies, typhoid , And vaccines directed against protection against pertussis. The combinations can be administered, for example, either as a mixture simultaneously, separately but simultaneously or in parallel; or sequentially. This includes the presentation that the combined agents are administered together as a therapeutic mixture, and the procedure where the combined agents are administered separately but simultaneously (eg, through separate intravenous lines to the same individual) Including. “Combination” administration further includes the separate administration of one of the compounds or agents given first, followed by the second.

  The albumin fusion proteins and / or polynucleotides of the invention can be administered alone or in combination with other therapeutic agents. Albumin fusion proteins and / or polynucleotide agents that can be administered in combination with the albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to: chemotherapeutic agents, antibiotics, steroidal and Non-steroidal anti-inflammatory agents, conventional immunotherapeutic agents, and / or therapeutic treatments described below. The combinations can be administered, for example, either as a mixture simultaneously, separately but simultaneously or in parallel; or sequentially. This includes instructions that the combined agents are administered together as a therapeutic mixture, and the procedure where the combined agents are administered separately but simultaneously (eg, through separate intravenous lines to the same individual). Also includes. “Combination” administration further includes the separate administration of one of the compounds or agents given first, followed by the second.

In one embodiment, the albumin fusion protein and / or polynucleotide of the invention is administered in combination with an anticoagulant. Anticoagulants that can be administered with the compositions of the present invention include, but are not limited to: heparin, low molecular weight heparin, warfarin sodium (eg, CUMADIN®), dicoumarol, 4- Hydroxycoumarin, anisindione (eg, MIRADON ^™ ), acenocoumarol (eg, nikumarone, SINTHROME ^™ ), indane-1,3-dione, fenprocoumone (eg, MARCUMA ^™ ), ethyl biscoumacetate (Eg TROMEXAN ^™ ), and aspirin. In certain embodiments, the compositions of the invention are administered in combination with heparin and / or warfarin. In another specific embodiment, the composition of the invention is administered in combination with warfarin. In another specific embodiment, the composition of the invention is administered in combination with warfarin and aspirin. In another specific embodiment, the composition of the invention is administered in combination with heparin. In another embodiment, the composition of the invention is administered in combination with heparin and aspirin.

In another embodiment, the albumin fusion protein and / or polynucleotide of the invention is administered in combination with a thrombolytic drug. Thrombolytic drugs that can be administered in combination with the compositions of the present invention include, but are not limited to: plasminogen, lys-plasminogen, α2-antiplasmin, streptokinase (eg, KABIKINASE ^™ ), Antiresplasase (eg, EMINASE ^™ ), tissue plasminogen activator (t-PA, artevase, ACTIVASE ^™ ), urokinase (eg, ABBO KINASE ^™ ), saurplase (prourokinase, single chain) Urokinase), and aminocaproic acid (eg AMICAR ^™ ). In certain embodiments, the compositions of the invention are administered in combination with a tissue plasminogen activator and aspirin.

In another embodiment, the albumin fusion protein and / or polynucleotide of the invention is administered in combination with an antiplatelet drug. Antiplatelet drugs that can be administered in combination with the compositions of the present invention include, but are not limited to, aspirin, dipyridamole (eg, PERSANTINE ^™ ), and ticlopidine (eg, TICLID ^™ ).

  In certain embodiments, the use of an anticoagulant, thrombolytic and / or antiplatelet drug in combination with the albumin fusion protein and / or polynucleotide of the present invention is thrombosis, arterial thrombosis, venous thrombosis, thromboembolism , Pulmonary embolism, atherosclerosis, myocardial infarction, transient cerebral ischemia (anemia) attack, disturbed unstable angina, diagnosis and / or treatment. In certain embodiments, the use of anticoagulant, thrombolytic and / or antiplatelet drugs in combination with the albumin fusion proteins and / or polynucleotides of the present invention is a risk of perianal thrombosis that may involve an angioplasty procedure To reduce the risk of stroke in patients with arterial fibrosis, including non-rheumatic arterial fibrosis, to reduce the risk of embolism associated with artificial heart valve and / or mitral valve disease To reduce the risk, it is contemplated for occult bleeding of the underlying graft. Other uses for the treatment of the present invention alone or in combination with antiplatelet drugs, anticoagulants and / or thrombolytic drugs include extracorporeal devices (eg, intravascular cannulas, vascular access in hemodialysis patients) Shouts, hemodialysis devices, and cardiopulmonary bypass devices), but not limited to.

In certain embodiments, an albumin fusion protein and / or polynucleotide of the invention comprises an antiretroviral agent, a nucleoside / nucleotide reverse transcriptase inhibitor (NRTI), a non-nucleoside reverse transcriptase inhibitor (NNRTI), and / or a protease inhibitor. Administered in combination with (PI). NRTIs that can be administered in combination with albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to: RETROVIR ^™ (zidovudine / AZT), VIDEOX ^™ (didanocin / ddI), HIVID ^™. (Zarcitabine / ddC), ZERIT ^™ (stavudine / d4T), EPIVIR ^™ (lamivudine / 3TC), and COMBIVIR ^™ (zidovudine / lamivudine). NNRTIs that can be administered in combination with the albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to: VIRAMUNE ^™ (Nevirapin), RESCRIPTOR ^™ (Delavirudine), and SUSTIVA ^™ (Efavirenz) . Protease inhibitors that can be administered in combination with albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to: CRIXIVAN ^™ (indinavir), NORVIR ^™ (ritonavir). )), INVIRASE ^™ (saquinavir)), and VIRACEPT ^™ (nelfinavir). In certain embodiments, the antiretroviral agent, nucleoside reverse transcriptase inhibitor, non-nucleoside reverse transcriptase inhibitor, and / or protease inhibitor is used to treat AIDS and / or to prevent or treat HIV infection. Any combination with the albumin fusion protein and / or polynucleotide of the present invention may be used.

Further NRTIs include: LODENOSINE ^™ (F-ddA; acid-stable adenosine NRTI; Triangle / Abbott; COVIRACIL ^™ (entricitabine / FTC; structurally related to lamivudine (3TC) But has 3-10 times greater activity in vitro; Triangle / Abbott); dOTC (BCH-1065 is also structurally related to lamivudine, but relative to a substantial proportion of lamivudine-resistant isolates Biochem Pharma); Adefovir (FDA rejected authorization for anti-HIV treatment; Gilad Sciences); PREVEON® (Ade) OVIR dipivoxil, active prodrug of adefovir (adefovir); its active form is PMEA-pp); TENOFOVIR ^TM (bis-POC PMPA, PMPA prodrugs; Gilead); DAPD / DXG (active metabolite of DAPD; Triangle / Abbott); D-D4FC (associated with 3TC and having activity against AZT / 3TC-resistant virus); GW420867X (Glaxo Wellcome); ZIAGEN ^™ (abacavir / 159U89; GlaxoIncCS; 87 (3 ′ azido-2 ′, 3′-dideoxyuridine; WO 99/66936); and β-L-FD4C containing S-acyl-2-thioethyl (SATE) And β-L-Fd
Prodrug form of dC (WO 98/17281).

Additional NNRTIs include the following: COACTINON ^™ (Emvirine / MKC-442, HEPT class potent NNRTI; Triangle / Abbott); CAPRAVILINE ^™ (AG-1549 / S-1153, activity against viruses including K103N variants Next generation NNRTI with Agro; Agouron); PNU-142721 (having 20-50 times greater activity than the precursor and active against K103N mutant; Pharmacia &Upjohn); DPC-961 And DPC-963 (a second generation derivative of efavirenz, designed to be active against viruses with the K103N mutation; DuPont); W-420867X (having 25-fold greater activity than HBY097 and active against the K103N mutant; Glaxo Wellcome); CALANOLIDE A (a naturally occurring drug from latex trees; any of the Y181C and K103N mutations Active against viruses containing both or both); and Propolis (WO 99/49830).

Additional protease inhibitors include the ^{following: LOPINAVI TM (ABT378 / r;} Abbott Laboratories); BMS-232632 ( ^{azapeptide; Bristol-Myres Squibb); TIPRANAVIR} TM (PNU-140690, a non-pepsin dihydropyrone; Pharmacia & Upjohn ); PD-178390 (non-peptidic dihydropyrone; Parke-Davis); BMS 232632 (azapeptide; Bristol-Myers Squibb); L-756, 423 (indinavir analog; Merck); DMP-450 ( Cyclic urea compounds; Avid &DuPont); AG-1776 (protease inhibitor Peptidomimetics have in vitro activity against resistant viruses; Agouron); VX-175 / GW-433908 phosphate prodrug of (amprenavir (amprenavir); Vertex & Glaxo Welcome ); CGP61755 (Ciba); and Agenerase ^TM (amp Lenavir; Glaxo Wellcome Inc.).

  Additional antiretroviral agents include fusion inhibitors / gp41 binding agents. Fusion inhibitors / gp41 binding agents include: T-20 (a peptide from residues 643-678 of the HIV gp41 transmembrane protein ectodomain that binds gp41 in the resting phase and transforms to the spindle cell state Trimeris) as well as T-1249 (second generation fusion inhibitor; Trimeris).

  Additional antiretroviral agents include fusion inhibitors / chemokine receptor antagonists. Fusion inhibitors / chemokine receptor antagonists include: CXCR4 antagonists (eg, AMD 3100 (bicyclam)), SDF-1 and its analogs, and ALX40-4C (cationic peptide), T22 (18) Trimeris) and T22 analogs T134 and T140; CCR5 antagonists (eg, RANTES (9-68), AOP-RANTES, NNY-RANTES, and TAK-779); and CCR5 / CXCR4 antagonists (eg, NSC 651016 ( Distamycin analog)). Also included are CCR2B, CCR3, and CCR6 antagonists. Chemokine receptor agonists (eg, RANTES, SDF-1, MIP-1α, MIP-1β, etc.) can also inhibit fusion.

Additional antiretroviral agents include integrase inhibitors. Integrase inhibitors include the following: dicaffeoylquinic acid (DFQA); L-ticolinic acid (dicaffeoyl tartaric acid (DCTA)); quinalizarin (QLC) and related anthraquinones; ZINTEVIR ^™ (AR 177, true Oligonucleotides that probably act on the cell surface but not integrase inhibitors; Arondex); as well as naphthol (eg, naphthol described in WO 98/50347).

Additional antiretroviral agents include: hydroxyurea-like compounds (eg, BCX-34 (purine nucleoside phosphorylase inhibitor; Biocrist); ribonucleotide reductase inhibitors (eg, DIDOX ^™ (Molecules for Health)); inosine mono Phosphate dehydrogenase (IMPDH) inhibitors (eg, VX-497 (Vertex)); and mycophoric acid (eg, CellCept (mycophenolate mofetil; Roche).

  Additional antiretroviral agents include: inhibitors of viral integrase, inhibitors of viral genome nuclear translocation (eg, arylene bis (methylketone) compounds); inhibitors of HIV entry (eg, AOP-RANTES, NNY- RANTES, RANTES-IgG fusion protein, RANTES soluble complex and glycosaminoglycan (GAG), and AMD-3100; nucleocapsid zinc finger inhibitors (eg, dithian compounds; HIV Tat and Rev targets; and pharmaceutical enhancers ( For example, ABT-378)).

Other antiretroviral and adjuvant treatments include: cytokines and lymphokines (eg, MIP-1α, MIP-1β, SDF-1α, IL-2, PROLEUKIN ^™ (aldesleukin / L2-7001; Chiron), IL-4, IL-10, IL-12, and IL-13); interferons (eg, IFN-α2a; TNFs, NFkB, GM-CSF, M-CSF, and IL-10) antagonist); agents that modulate immune activity (e.g., cyclosporine and prednisone (prednisone)); vaccines ^{(e.g., Remune TM (HIV Immunogen),} APL 400-003 (Apollon), recombinant gp120 and Furagume A bivalent (B / E) recombinant envelope glycoprotein, rgp120CM235, MN rgp120, SF-2rgp120, gp120 / soluble CD4 complex, Delta JR-FL protein, branched synthetic peptide derived from discontinuous gp120 C3 / C4 domain, Fusion component immunogens, and Gag, Pol, Nef, and Tat vaccines); gene-based therapeutics (eg, gene suppression elements (GSE; WO 98/54366), and intrakines (surface synthesis of newly synthesized CCR5) Generally modified CC chemokines (Yang et al., PNAS 94: 11567-72 (1997); Chen et al., Nat. Med. 3: 1110-16 (1997)); antibodies targeted against the ER to block (For example, anti-CXCR4 antibody 12G5, anti-CCR5 antibody 2D7, 5C7, PA8, PA9, PA10, PA11, PA12, and PA14, anti-CD4 antibody Q4120 and RPA-T4, anti-CCR3 antibody 7B11, anti-gp120 antibody 17b, 48d, 447-52D, 257-D, 268-D and 50.1, anti-Tat antibody, anti-TNF-α antibody, and monoclonal antibody 33A); aryl hydrocarbon (AH) receptor agonists and antagonists (eg, , TCDD, 3,3 ′, 4,4 ′, 5-pentachlorobiphenyl, 3,3 ′, 4,4′-tetrachlorobiphenyl, and α-naphthoflavone (WO 98/302
13)); as well as antioxidants (eg g-L-glutamyl-L-cysteine ethyl ester (g-GCE; WO 99/56764)).

  In further embodiments, the albumin fusion proteins and / or polynucleotides of the invention are administered in combination with an antiviral agent. Antiviral agents that can be administered with the albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to, acyclovir, ribavirin, amantadine, remantidine, maxamine, or thymalfasin. Not. In particular, the interferon albumin fusion protein can be administered in combination with any of these agents. Further, the interferon alpha albumin fusion protein can also be administered with any of these agents, and preferably the interferon alpha 2a or 2b albumin fusion protein is administered with any of these agents. In addition, the interferon beta albumin fusion protein can also be administered with any of these agents. Furthermore, any IFN hybrid albumin fusion protein can also be administered in combination with any of these agents.

  In the most preferred embodiment, the interferon albumin fusion protein is administered in combination with ribavirin. In a further preferred embodiment, the interferon alpha albumin fusion protein is administered in combination with ribavirin. In a further preferred embodiment, the interferon α2a albumin fusion protein is administered in combination with ribavirin. In a further preferred embodiment, the interferon α2b albumin fusion protein is administered in combination with ribavirin. In a further preferred embodiment, the interferon beta albumin fusion protein is administered in combination with ribavirin. In a further preferred embodiment, the hybrid interferon albumin fusion protein is administered in combination with ribavirin.

In other embodiments, the albumin fusion proteins and / or polynucleotides of the invention can be administered in combination with an anti-opportunistic infection agent. Anti-opportunistic infectious agents that can be administered in combination with the albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to: TRIMETHOPRIM-SULFAMETOXAZOLE ^™ , DAPSONE ^™ , PENTAMIDINE ^™ , ATOVAQUINE ^™ , ^{ISONIAZID TM, RIFAMPIN TM, PYRAZINAMIDE TM} , ETHAMBUTOL TM, RIFABUTIN TM, CLARITHROMYCIN TM, AZITHROMYCIN TM, GANCICLOVIR TM, FOSCARNET TM, CIDOFOVIR TM, FLUCONAZOLE TM, ITRACONAZOLE TM, KETOCONAZOLE ^{M, ACYCLOVIR TM, FAMCICOLVIR TM,} PYRIMETHAMINE TM, LEUCOVORIN TM, NEUPOGEN TM ( Filgrastim (filgrastim) / G-CSF) , and LEUKINE ^TM (sargramostim (sargramostim) / GM-CSF) . In certain embodiments, the albumin fusion proteins and / or polynucleotides of the present invention, opportunistic Pneumocystis carinii pneumonia infection to prophylactically treat or ^{prevent, TRIMETHOPRIM-SULFAMETHOXAZOLE TM, DAPSONE TM} , PENTAMIDINE TM, and / or ATOVAQUONE Used in any combination with ^TM . In another specific embodiment, the albumin fusion proteins and / or polynucleotides of the present invention may be used to prevent or treat opportunistic Mycobacterium avium complex infections with ISONIAZID ^™ , RIFAMPIN ^™ , PYRAZINAMIDE ^™ , and / or ETHAMBUTOL. Used in any combination with ^TM . In another specific embodiment, the albumin fusion protein and / or polynucleotide of the present invention is any of RIFABUTIN ^™ , CLARITHROMYCIN ^™ , and / or AZITHROMYCIN ^™ , for prophylactic treatment or prevention of opportunistic Mycobacterium tuberculosis infections. Used in combination. In another specific embodiment, the albumin fusion proteins and / or polynucleotides of the invention may be used with GANICLOVIR ^™ , FOSCARNET ^™ , and / or CIDOFOVIR ^™ to prevent or treat opportunistic cytomegalovirus infections prophylactically. Used in any combination. In another specific embodiment, the albumin fusion protein and / or polynucleotide of the present invention may be any of FLUCONAZOLE ^™ , ITRACONAZOLE ^™ , and / or KETOCONAZOLE ^™ to prevent or treat opportunistic fungal infections prophylactically. Used in combination. In another specific embodiment, the albumin fusion protein and / or polynucleotide of the present invention provides ACYCLOVIR ^™ and / or FAMCICOLVIR for prophylactic treatment or prevention of opportunistic herpes simplex virus type I and / or type II infection. Used in any combination with ^TM . In another specific embodiment, the albumin fusion proteins and / or polynucleotides of the invention are used in any combination with PYRIMETHAMINE ^™ and / or LEUCOVORIN ^™ to prevent or treat opportunistic Toxoplasma gondii infections prophylactically. Is done. In another specific embodiment, the albumin fusion proteins and / or polynucleotides of the invention are used in any combination with LEUCOVORIN ^™ and / or NEPOGEN ^™ to prevent or treat opportunistic bacterial infections prophylactically. The

  In a further embodiment, the albumin fusion protein and / or polynucleotide of the invention is administered in combination with an antibiotic. Antibiotics that can be administered with the albumin fusion protein and / or polynucleotide of the present invention include amoxicillin, β-lactamase, aminoglycoside, β-lactam (glycopeptide), β-lactamase, clindamycin, chloramphenicol, cephalo Examples include sporin, ciprofloxacin, erythromycin, fluoroquinolone, macrolide, metronidazole, penicillin, quinolone, rapamycin, rifampin, streptomycin, sulfonamide, tetracycline, trimethoprim, trimethoprim-sulfamethoxazole (sulfamthoxazole), and vancomycin However, it is not limited to these.

In other embodiments, the albumin fusion proteins and / or polynucleotides of the invention are administered in combination with an immunostimulatory agent. Immunostimulatory agents that can be administered in combination with the albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to: levamizole (eg, ERGAMISOL ^™ ), isoprinosine (eg, , INOSIPLEX ^™ ), interferon (eg, interferon α), and interleukin (eg, IL-2).

In other embodiments, the albumin fusion proteins and / or polynucleotides of the invention are administered in combination with an immunosuppressive agent. Immunosuppressive agents that can be administered in combination with the albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to: steroids, cyclosporine, cyclosporine analogs, cyclophosphamide methylprednisone, prednisone, Azathioprine, FK-506, 15-deoxyspergualin, and other immunosuppressive agents that act by inhibiting the functioning of responding T cells. Other immunosuppressive agents that can be administered in combination with the albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to: prednisolone, methotrexate, thalidomide, methoxalene, rapamycin, leflunomide, mizoribine (BREDININ ^TM), brequinar, deoxyspergualin, and Azasupiran (azaspirane) (SKF 105685), ORTHOCLONE OKT ( ^{R) 3 (muromonab-CD3),} SANDIMMUNE TM, NEORAL TM, SANGDYA TM ( cyclosporine), PROGRAF ( (Registered trademark) (FK506, tacrolimus), CELLCEP (R) (mycophenolate mofetil (mycophenolate Motefil), its active metabolite is mycophenolic acid), IMURAN ^TM (azathioprine), glucocorticosteroids, adrenocortical steroids (e.g., DELTASONE ^TM (prednisone) And HYDELTRASOL ^™ (prednisolone)), FOLEX ^™ and MEXATE ^™ (methotrexate), OXSORREN-ULTRA ^™ (methotrexalene) and RAPAMUNE ^™ (sirolimus). In certain embodiments, immunosuppressive agents may be used to prevent organ transplantation or bone marrow transplant rejection.

In further embodiments, the albumin fusion proteins and / or polynucleotides of the invention are administered alone or in combination with one or more intravenous immunoglobulin preparations. Intravenous immunoglobulin preparations that can be administered with the albumin fusion proteins and / or polynucleotides of the present invention include GAMMAR ^™ , IVEEGAM ^™ , SANDOLOBULIN ^™ , GAMMAGARD S / D ^™ ATGAM ^™ (anti-thymocyte globulin) and Examples include, but are not limited to GAMIMYNE ^™ . In certain embodiments, albumin fusion proteins and / or polynucleotides of the invention are administered in combination with intravenous immunoglobulin preparations in transplantation therapy (eg, bone marrow transplantation).

  In another embodiment, the albumin fusion proteins and / or polynucleotides of the present invention are administered alone or as part of a combination therapy for the treatment of cancer, either in vivo to a patient or in vitro to a cell. Is done. In certain embodiments, albumin fusion proteins, particularly IL-2-albumin fusions, are passive immunotherapy for cancer (see, eg, Science Express at Dudley et al., Www.scienceexpress.org, September 19, 2002 ( This whole is now repeatedly administered during adoptive cell metastasis treatment for metastatic melanoma as described in (incorporated herein by reference).

  In certain embodiments, the albumin fusion proteins and / or polynucleotides of the invention are administered alone or in combination with an anti-inflammatory agent. Anti-inflammatory agents that can be administered in combination with albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to: corticosteroids (eg, betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, Methylprednisolone, prednisolone, prednisone and triamcinolone), non-steroidal anti-inflammatory drugs (eg, diclofenac diflunisal, etodolac, fenoprofen, fructaphenine, flurbiflofen, ibuprofen, indomethacin, ketoprofen, meclofenamate (meclofenate) ), Mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin, fu Nilbutazone, piroxicam, sulindac, tenoxicam, thiaprofenic acid, and tolmethine), and antihistamines, aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles, pyrazolones, salicylic acid derivatives, Thiazinecarboxamide, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine, bucolome, difenpyramide, ditazole, emorphazone, guaiazulene, nabumetone, nimesulide (Nimesulide), orgotein, oxaceptrol, paraniri Paraline, perisoxal, pifoxime, proquazone, proxazole, and tenidap.

  In further embodiments, the compositions of the invention are administered alone or in combination with an anti-angiogenic agent. Anti-angiogenic agents that can be administered in combination with the compositions of the present invention include, but are not limited to: Angiostatin (Entremed, Rockville, MD), Troponin-1 ( (Boston Life Sciences, Boston, MA), anti-invasive factor, retinoic acid and its derivatives, paclitaxel (Taxol), suramin, tissue inhibitor of metalloproteinase-1, metalloproteinase-2 tissue inhibitor, VEGI, plasminogen activator Inhibitor-1, plasminogen activator inhibitor-2 and various forms of lighter “d group” transition metals.

  Lighter “d group” transition metals include vanadium, molybdenum, tungsten, titanium, niobium and tantalum species, for example. Such transition metal species can form transition metal complexes. Suitable complexes of the above transition metal species include oxo transition metal complexes.

  Representative examples of vanadium complexes include oxo vanadium complexes (eg, vanadic acid complexes and vanadyl complexes). Suitable vanadate complexes include metavanadate and orthovanadate complexes (eg, ammonium metavanadate, sodium metavanadate and sodium orthovanadate). Suitable vanadyl complexes include, for example, vanadyl sulfate, including vanadyl acetylacetonate and vanadyl sulfate hydrates such as vanadyl sulfate monohydrate and vanadyl sulfate trihydrate.

Representative examples of tungsten and molybdenum complexes also include oxo complexes. Suitable oxo tungsten complexes include tungstic acid and tungsten oxide complexes. Suitable tungstate acid complexes include ammonium tungstate, calcium tungstate, sodium tungstate dihydrate and tungstic acid. Suitable tungsten oxides include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxomolybdenum complexes include molybdate complexes, molybdenum oxide complexes and molybdenyl complexes.
Suitable molybdate complexes include ammonium molybdate and its hydrate, sodium molybdate and its hydrate, and potassium molybdate and its hydrate. Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum (VI) oxide and molybdic acid. Suitable molybdenyl complexes include, for example, molybdenyl acetylacetonate. Other suitable tungsten and molybdenum complexes include, for example, hydroxo derivatives derived from glycerol, tartaric acid and sugars.

  A wide variety of other anti-angiogenic factors may also be utilized in the context of the present invention. Representative examples include, but are not limited to: platelet factor 4; protamine sulfate; sulfated chitin derivatives (prepared from queen crab shells) (Murata et al., Cancer Res. 51: 22-26, 1991); sulfated polysaccharide peptidoglycan complex (SP-PG) (the function of this compound can be enhanced by the presence of steroids (eg estrogen) and tamoxifen citrate); staurosporine; Modulators of substrate metabolism (including, for example, proline analogs, cishydroxyproline, d, L-3,4-dehydroproline, thiaproline, α, α-dipyridyl, aminopropionitrile fumarate); 4-propyl-5- ( 4-pyridinyl) -2 (3H) -oxazolone Methotrexate; mitoxantrone; heparin; interferon; 2 macroglobulin-serum; ChIMP-3 (Pavloff et al., J. Bio. Chem. 267: 17321-17326, 1992); chymostatin (Tomkinson et al., Biochem J. 286: 475). 480, 1992); cyclodextrin tetradecasulfate; eponemycin; camptothecin; fumagillin (Ingber et al., Nature 348: 555-557, 1990); gold sodium thiomalate ("GST"; Matsubara and Ziff, Jiff, Clin. Invest. 79: 1440-1446, 1987); anti-collagenase-serum; olmes et al., J. Biol. Chem. 262 (4): 1659-1664, 1987); Bisantrene (National Cancer Institute); Robenzalite disodium (N- (2) -carboxyphenyl-4-chloroanthronyl). Disodium acid, or “CCA”; Takeuchi et al., Agents Actions 36: 312-316, 1992); and metalloproteinase inhibitors (eg, BB94).

  Additional anti-angiogenic factors that may also be utilized in the context of the present invention include thalidomide (Celgene, Warren, NJ); vasodilatory steroids; AGM-1470 (H. Brem and J. Folkman J Pediatr. Surg. 28. : 445-51 (1993)); integrin αvβ3 antagonist (C. Storgard et al., J Clin. Invest. 103: 47-54 (1999)); carboxyaminoimidazolide; (Institute, Bethesda, MD); Combrestatin A-4 (CA4P) (OXiGENE, Boston, MA) Squalamine (Magnaine Pharmaceuticals, Plymouth Meeting, PA); TNP-470, (Tap Pharmaceuticals, Deerfield, IL); ZD-0101 AstraZeneca (London, UK) B25; 41251 (PKC 412); CM101; Dexrazoxane (ICRF187); DMXAA; Endostatin; Flavopridiol; Genestein; GTE; ImmTher (ZD1839); Octreotide (Somatostatin); int; PI-88; Prinomastat (AG-3340) Purlytin; Suradista (FCE26644); Tamoxifen (Nolvadex); Tazarotene; tetrathiomolybdate; Xeloda (Capecitabine); and 5-fluorouracil.

  Anti-angiogenic agents that can be administered in combination with a compound of the present invention can act through a variety of mechanisms, including but not limited to: Proteolytic degradation of extracellular matrix Inhibition, blocking the function of endothelial cell-extracellular matrix adhesion molecules by antagonizing the function of angiogenic factors such as growth factors, and inhibition of integrin receptors expressed in proliferating endothelial cells. Examples of anti-angiogenesis inhibitors that interfere with extracellular matrix proteolysis and can be administered in combination with the compositions of the present invention include, but are not limited to: AG-3340 (Agouron, La Jolla, CA), BAY-12-9566 (Bayer, West Haven, CT), BMS-275291 (Bristol Myers Squibb, Princeton, NJ), CGS-27032A (Novatis, East Hanover, NJst) , UK) and Metastat (Aeterna, St-Foy, Quebec). Examples of anti-angiogenesis inhibitors that act by blocking the function of endothelial cell-extracellular matrix adhesion molecules and that can be administered in combination with the compositions of the present invention include, but are not limited to: Not: EMD-121974 (Merck KcgaA Darmstadt, Germany) and Vitaxin (Ixsys, La Jolla, CA / Medimune, Gaithersburg, MD). Examples of anti-angiogenic agents that act by directly antagonizing or inhibiting this angiogenic factor and can be administered in combination with the compositions of the present invention include the following: , But not limited to: Angiozyme (Ribozyme, Boulder, CO), anti-VEGF antibody (Genentech, S. San Francisco, Calif.), PTK-787 / ZK-225846 (Novartis, Basel, Switzerland, Sugen 101), SU-101 (SU-101) S. San Francisco, CA), SU-5416 (Sugen / Pharmacia Upjohn, Bridgewater, NJ), and SU-6668 (Sugen). Other anti-angiogenic agents act to indirectly inhibit angiogenesis. Examples of indirect inhibitors of angiogenesis that can be administered in combination with the compositions of the present invention include, but are not limited to: IM-862 (Cytran, Kirkland, WA), interferon-α, IL-12 (Roche, Nutley, NJ), and pentosan polysulfate (Georgetown University, Washington, DC).

  In certain embodiments, the use of a composition of the invention in combination with an anti-angiogenic agent is for the treatment, prevention, and / or amelioration of an autoimmune disease (eg, an autoimmune disease described herein). Is intended for.

  In certain embodiments, the use of the compositions of the invention in combination with an anti-angiogenic agent is contemplated for the treatment, prevention, and / or recovery of arthritis. In a more specific embodiment, the use of the composition of the invention in combination with an anti-angiogenic agent is contemplated for the treatment, prevention and / or recovery of rheumatoid arthritis.

  In another embodiment, a polynucleotide encoding a polypeptide of the invention is administered in combination with an angiogenic protein or a polynucleotide encoding an angiogenic protein. Examples of angiogenic proteins that can be administered with the compositions of the present invention include acidic and basic fibroblast growth factor, VEGF-1, VEGF-2, VEGF-3, epidermal growth factor α and epidermal growth factor β. Platelet-derived endothelial cell growth factor, platelet-derived growth factor, tumor necrosis factor α, hepatocyte growth factor, insulin-like growth factor, colony stimulating factor, macrophage colony stimulating factor, granulocyte / macrophage colony stimulating factor, and nitric oxide synthase However, it is not limited to these.

  In further embodiments, the compositions of the invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that can be administered with the therapeutic agents of the present invention include, but are not limited to: alkylating agents (eg, nitrogen mustard (eg, mechlorethamine, cyclophosphamide, cyclophosphamide ifosfamide ( Ifosfamide), melphalan (L-sarcolicine), and chlorambucil), ethyleneimines and methylmelamines (eg, hexamethylmelamine and thiotepa), alkylsulfonates (eg, busulfan), nitrosoureas (eg, carmustine (BCNU)) ), Lomustine (CCNU), semustine (methyl-CCNU), and streptozocin (streptozotocin), triazenes (eg, dacarbazine (DTIC)) Dimethyltriazenoimidazole carboxamide)), folic acid analogs (eg, methotrexate (amethopterin)), pyrimidine analogs (eg, fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorodeoxyuridine; FudR)), and Cytarabine (cytosine arabinoside)), purine analogs and related inhibitors (eg, mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG), and pentostatin (2′-deoxycoformycin) (Deoxycoformin))), vinca alkaloids (eg, vinblastine (VLB, vinblastine sulfate)) and vincristine (vincristin sulfate)), Epipodophyllotoxins (eg, etoposide and teniposide), antibiotics (eg, dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, primycin (mitromycin), and mitomycin (mitomycin C) ), Enzymes (eg, L-asparaginase), biological response modifiers (eg, interferon-α and interferon-α-2b), platinum coordination compounds (eg, cisplatin (cis-DDP) and carboplatin), anthracenedione (Mitoxantrone), substituted urea (eg, hydroxyurea), methylhydrazine derivatives (eg, procarbazine (N-methylhydrazine; MIH), corticosteroids (eg, predo) Nizone), progestins (eg, hydroxyprogesterone caproate, medroxyprogesterone, medroxyprogesterone acetate, and megestrol acetate), estrogens (eg, diethylstilbestrol (DES), diethylstilbestrol diphosphate, estradiol, and Ethinylestradiol), antiestrogens (eg tamoxifen), androgens (testosterone proprionate and fluoxymesterone), antiandrogens (eg flutamide), gonadotropin releasing hormone analogs (eg leuprolide), Other hormones and hormone analogs (eg methyltestosterone, estramustine, Sodium Toramusuchinrin acid, chlorotrianisene Nisen, and testolactone), and others (e.g., Jikarubajin, glutamic acid, and mitotane).

In one embodiment, the compositions of the invention may be administered in combination with one or more of the following drugs: infliximab (also known as Remicade ^™ Centocor, Inc.), Trocade (Raoche, RO-33-3555), Leflunomide (also known as Arava ^{™ from} Hoechst Marion Roussel), Kineret ^™ (IL-1 receptor antagonist, also known as Anakinra from Amgen, Inc.).

  In certain embodiments, the compositions of the invention are administered in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) or in combination with one or more components of CHOP. In one embodiment, the composition of the invention is administered in combination with an anti-CD-20 antibody (human monoclonal anti-CD20 antibody). In another embodiment, the composition of the invention is administered in combination with an anti-CD20 antibody and CHOP, or of one or more components of a CDOP antibody and CHOP (specific cyclophosphamide and / or prednisone). Administered in any combination. In certain embodiments, the compositions of the invention are administered in combination with Rituximab. In further embodiments, the compositions of the invention are administered with Rituxmab and CHOP or with any combination of one or more components of Rituxmab and CHOP, specifically cyclophosphamide and / or prednisone. In certain embodiments, the compositions of the invention are administered in combination with tositomab. In further embodiments, the compositions of the invention are administered with tositomamab and CHOP, or with any combination of one or more components of tositomab and CHOP, specifically cyclophosphamide and / or prednisone. The anti-CD20 antibody may bind to a radioisotope, toxin or cytotoxic drug as required.

In another specific embodiment, the compositions of the invention are administered in combination with Zevalin ^™ . In further embodiments, the compositions of the invention are administered with Zevalin ^™ and CHOP, or with any combination of one or more of the components of Zevalin ^™ and CHOP, specifically cyclophosphamide and / or prednisone. .
Zevalin ^™ can bind to one or more radioisotopes. Particularly preferred isotopes are ⁹⁰ Y and ¹¹¹ In.

  In further embodiments, the albumin fusion proteins and / or polynucleotides of the invention are administered in combination with cytokines. Cytokines that can be administered with albumin fusion proteins and / or polynucleotides of the invention include IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-γ and TNF- Although α is mentioned, it is not limited to these. In another embodiment, the albumin fusion protein and / or polynucleotide of the present invention is any interleukin (IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL- 6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, Including, but not limited to IL-19, IL-20 and IL-21).

  In one embodiment, the albumin fusion proteins and / or polynucleotides of the invention are administered in combination with a TNF family member. TNF molecules, TNF-related molecules or TNF-like molecules that can be administered with the albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to: soluble forms of TNF-α, lymphotoxin-α ( LT-α, also known as TNF-β), LT-β (found in complex heterotrimers LT-α2-β), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF- γ (International Publication No. WO96 / 14328), AIM-I (International Publication No. WO97 / 33899), Endokine-α (International Publication No. WO98 / 07880), OPG, and Neutrokine-α (International Publication No. WO98 / 18921) , OX40, and nerve growth factor (NGF), Rabi and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2 (International Publication No. WO96 / 34095), DR3 (International Publication No. WO97 / 33904), DR4 (International Publication No. WO98 / 32856), TR5 (International Publication number WO 98/30693), TRNK, TR9 (international publication number WO 98/56892), TR10 (international publication number WO 98/54202), 312C2 (international publication number WO 98/06842), and TR12, and soluble forms of CD154, CD70, And CD153.

  In further embodiments, the albumin fusion proteins and / or polynucleotides of the invention are administered in combination with an angiogenic protein. Angiogenic proteins that can be administered with the albumin fusion proteins and / or polynucleotides of the present invention include the glioma-derived growth factor (GDGF) as disclosed in European Patent EP-399816; Europe Platelet-derived growth factor-A (PDGF-A) as disclosed in patent EP-682110; Platelet-induced growth factor-B (PDGF-B) as disclosed in European patent EP-282317; International Publication No. WO92 / Placental growth factor (PIGF) as disclosed in 06194; Placental growth factor-2 (PIGF-2) as disclosed in Hauser et al., Gorwt Factors 4: 259-268 (1993); International Publication No. WO90 / 13649 Vascular endothelial growth factor (VEGF) such as: Vascular endothelial growth factor-A (VEGF-A) as disclosed in European Patent EP-506477; Vascular endothelial growth factor as disclosed in International Publication No. WO 96/39515 -2 (VEGF-2); Vascular Endothelial Growth Factor-B (VEGF-3); Vascular Endothelial Growth Factor B-186 (VEGF-B186) as disclosed in International Publication No. WO 96/26736; International Publication No. WO 98 Vascular endothelial growth factor-D (VEGF-D) as disclosed in US / 02543; Vascular endothelial growth factor-D (VEGF-D) as disclosed in WO 98/07832; and German patent Examples include, but are not limited to, vascular endothelial growth factor-E (VEGF-E) as disclosed in DE19639601. The above references are incorporated herein by reference in their entirety.

  In further embodiments, the albumin fusion proteins and / or polynucleotides of the invention are administered in combination with fibroblast growth factor. Examples of fibroblast growth factors that can be administered with the albumin fusion protein and / or polynucleotide of the present invention include FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, and FGF-7. FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15.

In a further embodiment, the albumin fusion protein and / or polynucleotide of the invention is administered in combination with a hematopoietic growth factor. Hematopoietic growth factors that can be administered with the albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to: granulocyte macrophage colony stimulating factor (GM-CSF) (sargramostim, LEUKINE ^™ ). , PROKINE ^™ ), granulocyte colony-stimulating factor (G-CSF) (filgrastim, NEPOGEN ^™ ), macrophage colony-stimulating factor (M-CSF, CSF-1) erythropoietin (epoetin α, EPOGEN ^™) PROCRIT ^™ ), stem cell factor (SCF, c-kit ligand, steel factor), megakaryocyte colony stimulating factor, PIXY321 (GMCSF / IL-3) Fusion protein), interleukins (especially any one or more of IL-1 to IL-12), interferon gamma, or thrombopoietin.

  In certain embodiments, the albumin fusion proteins and / or polynucleotides of the invention comprise an adrenergic blocker (eg, acebutolol, atenolol, betaxolol, bisoprolol, carteolol, labetalol, metoprolol, nadolol, oxprenolol, penbutolol, Administered in combination with pindolol, propranolol, sotalol, and timolol.

  In another embodiment, the albumin fusion protein and / or polynucleotide of the present invention comprises an antiarrhythmic drug (eg, adenosine, amidoarone, bretylium, digitalis, digoxin, digitoxin, diliazem, disopyramide, esmolol, flecainide. , Lidocaine, mexiletine, moricidine, phenytoin, procainamide, N-acetylprocainamide, propaphenone, propranolol, quinidine, sotalol, tocainide, and verapamil).

In another embodiment, the albumin fusion protein and / or polynucleotide of the invention comprises a diuretic (eg, a carbonic anhydrase-inhibitor (eg, acetazolamide, dichlorphenamide, and metasolamide), an osmotic diuretic ( for example, glycerin, isosorbide, mannitol, and ^{urea), Na} + -K + -2Cl ^- diuretics that inhibit symport (e.g., furosemide, bumetanide, azosemide, piretanide, tripamide, ethacrynic acid, muzolimine, and torsemide), thiazide and Thiazide-like diuretics (eg, bendroflumethiazide, benzthiazide, chlorothiazide, hydrochlorothiazide, hydroflumethiazide, methiclotiazide, polythiazide, trichormethiazide (trichormet) iaside), chlorthalidone, indapamide, metolazone, and quinesazone), potassium-sparing diuretics (eg, amiloride and triamterene), and mineralocorticoid receptor antagonists (eg, spironolactone, canrenone, and potassium canrenoate) Be administered.

In one embodiment, the albumin fusion proteins and / or polynucleotides of the invention are administered in combination with treatment for endocrine and / or hormonal imbalance disorders. Treatments for endocrine and / or hormonal imbalance disorders include, but are not limited to: ¹²⁷ I, radioactive isotopes of iodine (eg, ¹³¹ I and ¹²³ I); recombinant growth hormone (eg, , HUMATROPE ^™ (recombinant somatropin); growth hormone analogs (eg PROTROPIN ^™ (somatrem)); dopamine agonists (eg PARLODEL ^™ (bromocriptine); somatostatin analogues (eg SANDOSTATIN ^™ (octreotide); gonadotropin preparations (eg PREGNYL ^™ , APL ^™ and PROFASI ^™ (chorionic gonadotropin (CG)), PERGONAL ^™ (menotropin), and METRODIN ^™ ( Urinary follicle stimulating hormone (uFSH)); synthetic human gonadotropin-releasing hormone preparations (eg, FACTREL ^™ and LUTREPULSE ^™ (gonadorelin hydrochloride); synthetic gonadotropin agonists (eg, LUPRON ^™ (leuprolide acetate), SUPPRELIN ^™ (histrellin acetate ( histellin)), SYNAREL ^™ (nafarelin acetate), and ZOLADEX ^™ (goserelin acetate); synthetic preparations of thyroid-stimulating hormone-releasing hormone (eg, RELEFACT TRH ^™ and THYPINENE ^™ (protilelin); recombinant human TSH (eg, THYROGEN) ^TM); synthesis preparation of the sodium salt of the natural isomers of thyroid hormones _(e.g., ^L-T 4 _TM, SYNTHROI ^TM and Levothroid ^TM (levothyroxine ^{_{sodium), L-T 3 TM,}} CYTOMEL TM and TRIOSTAT ^TM (Rio Ciro in sodium (liothyroine sodium)), and THYROLAR ^TM (liotrix)); antithyroid compounds (e.g., 6-n -Propylthiouracil (propylthiouracil), 1-methyl-2-mercaptoimidazole and TAPAZOLE ^™ (methimazole), NEO-MERCAZOLE ^™ (carbimazole); β-adrenergic receptor antagonists (eg propranolol and esmolol); Ca ²⁺ Channel blockers; dexamethasone and iodinated radiocontrast agents (eg, TELEPAQUE ^™ (iopanic acid (io panic acid)) and ORGRAFIN ^™ (sodium ipodate).

Additional treatments for endocrine and / or hormonal imbalance disorders include, but are not limited to: estrogen or conjugated estrogens (eg, ESTRACE ^™ (estradiol), ESTINYL ^™ (ethynylestradiol), PREMARIN ^™ , ESTRATAB, ^{TM, ORTHO-EST TM, oGEN} TM and estropipate (estropipate) (estrone), ESTROVIS ^TM (quinestrol), ESTRADERM ^TM (estradiol), DELESTROGEN ^TM and VALERGEN ^TM (estradiol valerate), DEPO-eSTRADIOL CYPIONATE ^TM and ESTROJECT LA ^TM (Saipion acid (cyp Onate) estradiol)); anti-estrogens (e.g., NOLVADEX ^TM (tamoxifen), SEROPHENE ^TM and Clomid ^TM (clomiphene); progestins (e.g., DURALUTIN ^TM (hydroxyprogesterone caproate), ^{MPA TM} and DEPO-Provera® ^TM (acetic medroxy Progesterone), PROVERA ^™ and CYCRIN ^™ (MPA), MEGACE ^™ (megestrol acetate), NORLUTIN ^™ (norethindrone), and NORLUTATE ^™ and AYGESTIN ^™ (norethindrone acetate) (eg, NORPLANT ^™ SYSTEM Subcutaneous implant)); Mon (e.g., RU ^{486 TM} (mifepristone); hormonal contraceptives (e.g., ENOVID ^TM (norethynodrel + mestranol), intrauterine device emitting PROGESTASERT ^{TM (progesterone), LOESTRIN TM, BREVICON TM,} MODICON TM, GENORA ^{TM, NELONA TM, NORINYL TM,} OVACON-35 TM and ^{OVACON-50 TM} (ethinyl estradiol / ^{norethindrone), LEVLEN TM, NORDETTE TM,} TRI-LEVLEN TM and ^{TRIPHASIL-21 TM} (ethinyl estradiol / levonorgestrel (levonorgestrel) LO / OVRAL ^TM and OVRAL ^TM (Ethini Le estradiol / norgestrel), DEMULEN ^TM (ethinyl estradiol / ethynodiol ^{diacetate), NORINYL TM, ORTHO-NOVUM} TM, NORETHIN TM, GENORA TM, and NELOVA ^TM (norethindrone / mestranol), DESOGEN ^TM and ORTHO-CEPT ^TM (ethinyl estradiol / OTHHO-CYCLEN ^™ and ORTHO-TRICYCLEN ^™ (ethynylestradiol / norgestimate), MICRONOR ^™ and NOR-QD ^™ (norethindrone), and OVRETTE ^™ (norgestrel)).

Additional treatments for endocrine and / or hormonal imbalance disorders include, but are not limited to: testosterone esters (eg, methenolone acetate and testosterone undecanoate; parenteral and oral androgens (eg, TESTOJECT) -50 ^™ (testosterone), TESTEX ^™ (testosterone propionate), DELATESTRYL ^™ (testosterone enanthate), DEPO-TESTOSTERONE ^™ (testosterone cypionate), DANOCRINE ^™ (danazol), HALOTESTIN ^™ (fluoxymesterone), THL ^TM, TESTRED ^TM and VIRILON ^TM (methyltestosterone), and OXA DRIN ^TM (oxandrolone (Oxandrolone)); testosterone transdermal system (e.g., TESTODERM ^TM); androgen receptor antagonists and 5-alpha-reductase inhibitors (e.g., ANDROCUR ^TM) (cyproterone acetate), EULEXIN ^TM (flutamide), and PROSCAR ^™ (finasteride); corticotropin preparations (eg, CORTROSYN ^™ (cosyntropin); corticosteroids and their synthetic analogs (eg, ACLOVATE ^™ ) (alclomethasone dipropionate), CYCLOCART ^™ (amsinonide), ^TM VANCERIL ^™ (beclomethasone dipropionate), CELESTONE ^™ (Betamethasone), BENISON ^™ and UTICORT ^™ (betamethasone benzoate), DIPROSON ^™ (betamethasone dipropionate), CELESTONE PHOSPHATE ^™ (sodium betamethasone phosphate), CELESTONE SOLUSPAN ^™ ET, betamethasone sodium acetate A ^TM and VALISONE ^TM (betamethasone valerate), TEMOVATE ^TM (clobetasol propionate), CLODERM ^TM (clocortolone pivalate), CORTEF ^TM and HYDROCORTONE ^TM (cortisol (hydrocortisone)), HYDROCORTONE aCETATE ^TM (acetate cortisol (hydrocortisone)), LOCOI ^TM (butyrate cortisol (hydrocortisone)), HYDROCORTONE PHOSPHATE ^TM (cortisol (hydrocortisone) sodium phosphate), A-HYDROCORT ^TM and SOLU CORTEF ^TM (cortisol (hydrocortisone) sodium succinate), WESTCORT ^TM (valerate cortisol (hydrocortisone)) , cORTISONE aCETATE ^TM (cortisone acetate), DESOWEN ^TM and TRIDESILON ^TM (desonide), TOPICORT ^TM (desoximetasone), DECADRON ^TM (dexamethasone), DECADRON LA ^TM (dexamethasone acetate), DECADRON PHOSPHATE ^TM and HEXADROL PHOSPHA E ^TM (dexamethasone sodium phosphate, FLORONE ^TM and MAXIFLOR ^TM (di diflorasone diacetate), FLORINEF ACETATE ^TM (fludrocortisone acetate), AEROBID ^TM and Nasalide ^TM (flunisolide), FLUONID ^TM and SYNALAR ^TM (fluocinolone acetonide) , LIDEX ^TM (fluocinonide), ^{FLUOR-OP TM} and ^{FML TM} (fluorometholone), CORDRAN ^TM (flurandrenolide), hALOG ^TM (halcinonide), HMS LIZUIFILM ^TM (medrysone), MEDROL ^TM (methylprednisolone), DEPO- MEDROL ^TM and MEDROL aCETATE ^TM (methyl acetate prednisone) A-METHAPRED ^TM and Solumedrol ^TM (sodium methyl prednisolone succinate), ELOCON ^TM (mometasone furoate ^{(memotasone furoate)), HALDRONE TM} ( acetic paramethasone), DELTA-CORTEF ^TM (prednisolone), ECONOPRED ^TM (prednisolone acetate), HYDELTRASOL ^™ (prednisolone sodium phosphate), HYDELTRA-T. B. A ^TM (prednisolone tebutate ^{(prednisolone tebutate)), DELTASONE TM} ( prednisone), Aristocort ^TM and KENACORT ^TM (triamcinolone (triamcinolone)), KENALOG ^TM (triamcinolone acetonide), Aristocort ^TM and KENACORT DIACETATE ^TM (triamcinolone longitudinals acetate) , And ARISTOSPAN ^™ (triamcinolone hexaacetonide); inhibitors of the biosynthesis and action of corticosteroids (eg CYTADREN ^™ (aminoglutethimide), NIZORAL ^™ (ketoconazole), MODRASSTANE ^™ (trirostane), and METOPIRON E ^TM (metyrapone); bovine, porcine or human insulin or mixtures thereof; insulin analogues; recombinant human insulin (e.g., HUMULIN ^TM and Novolin ^TM); oral hypoglycemic agents (e.g., ORAMIDE ^TM and ORINASE ^TM (tolbutamide), DIABINESE ^TM (chlorpropamide), TOLAMIDE ^TM and TOLINASE ^TM (tolazamide), DYMELOR ^TM (acetohexamide), ^{glibenclamide,} MICRONASE ^TM, DIBETA TM and GLYNASE ^TM (glyburide), Glucotrol ^TM (glipizide), and DIAMICRON ^TM (gliclazide ), GLUCOPHAGE ^TM (metformin), ciglitazone, pioglitazone And α- glucosidase inhibitors; bovine or porcine glucagon; somatostatin (e.g., Sandostatin ^TM (octreotide); and diazoxide (e.g., Proglycem ^TM (diazoxide)).

In one embodiment, the albumin fusion protein and / or polynucleotide of the invention is administered with a treatment for uterine motility disorder. Treatments for uterine motility disorders include, but are not limited to: estrogen drugs (eg, conjugated estrogens (eg, PREMARIN® and ESTRATAB®), estradiol (eg, CLIMARA) (Registered trademark) and ALOROR (registered trademark), estropipate, and chlorotrianicene); progestin drugs (eg, AMEN® (medroxyprogesterone), MICRONOR® (norethidrone acetate) )), PROMETRIUM® progesterone, and megestrol acetate); and estrogen / progesterone combination therapeutics (eg, conjugated) Estrogens / medroxyprogesterone (eg, PREMPRO ^™ and PREMPPHASE®) and norethindrone acetate / ethynyl estradiol (eg, FEMHRT ^™ ).

In further embodiments, the albumin fusion proteins and / or polynucleotides of the invention are administered in combination with a drug effective in treating iron deficiency and hypochromic anemia. These drugs include, but are not limited to, ferrous sulfate (ferrous sulfate, FEOSOL ^TM), ferrous fumarate (e.g., FEOSTAT ^TM), ferrous gluconate (e.g., FERGON ^TM ), Polysaccharide-iron complex (eg, NIFERREX ^™ ), iron dextran injection (eg, INFED ^™ ), cupric sulfate, pyroxidine, riboflavin, vitamin B ₁₂ , cyanocobalamin injection (eg, REDISOL ^™ , RUBRAMIN PC) ^TM ), hydroxocobalamin, folic acid (eg FOLVITE ^™ ), leucovorin (folinic acid, 5-CHOH4PteGlu, citrobolum factor) or WELLCOVORIN (calcium salt of leucovorin), transferrin or ferriti N.

  In certain embodiments, the albumin fusion proteins and / or polynucleotides of the invention are administered in combination with agents used to treat psychiatric disorders. Psychiatric drugs that can be administered with the albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to: antipsychotics (eg, chlorpromazine, chlorprothixene, clozapine, fluphenazine, Haloperidol, loxapine, mesoridazine, molindone, olanzapine, perphenazine, pimozide, quetiapine, risperidone, thioridazine, thiothixene, trifluoperazine, and triflupromazine), antidepressants (eg, carbamazepine, divalprox sodium (divalproex sodium), Lithium carbonate and lithium citrate), antidepressants (eg, amitriptyline, amoxapine, bupropion (Amfebutamon) ), Citalopram, clomipramine, desipramine, doxepin, fluvoxamine, fluoxetine, imipramine, isocarboxazide, maprotiline, mirtazapine, nefazodone, nortriptyline, paroxetine, phenelzine, protriptyline, sertraline, tranylcypromine, trazodone, phazodone Xin), anxiolytics (eg, alprazolam, buspirone, chlordiazepoxide, chlorazepate, diazepam, halazepam, lorazepam, oxazepam, and prazepam), and stimulants (eg, d-amphetamine, methylphenidate, and pemoline).

  In other embodiments, the albumin fusion proteins and / or polynucleotides of the present invention are administered in combination with agents used to treat neurological disorders. Neurological agents that can be administered with the albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to: antispasmodics (eg, carbamazepine, clonazepam, ethosuximide, phenobarbital, phenytoin, primidone, Valproic acid, divalprox sodium, felbamate, gabapentin, lamotrigine, levetiracetam, oxcarbazepine, tiagabine, topiramate, zonisamide, diazepam, lorazepam, and clonazepam), anti- tremor drugs (eg, levodopa / carbidopa Seresillin, amantidine, bromocriptine, pergolide, ropinirole, pramipexole, benztropine; biperidene; et Ropajin; procyclidine; trihexyphenidyl, tolcapone (tolcapone)), and ALS therapeutics (e.g., riluzole (riluzole)).

  In another embodiment, the albumin fusion protein and / or polynucleotide of the invention is administered in combination with a vasodilator and / or calcium channel blocker. Vasodilators that can be administered with the albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to: angiotensin converting enzyme inhibitor (ACE) inhibitors (eg, papaverine, isoxsuprin, benazepril, captopril). , Cilazapril, enalapril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, spirapril, trandolapril, and nilidrin) and nitrates (eg, isosorbide dinitrate, isosorbide mononitrate, and nitroglycerin). Examples of calcium channel blocking agents that can be administered in combination with the albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to, amlodipine, bepridil, felodipine, flunarizine, isradipine, nicardipine, nimodipine, and verapamil. Not.

In certain embodiments, the albumin fusion proteins and / or polynucleotides of the invention are administered in combination with treatment for gastrointestinal disorders. Gastrointestinal disorder treatment agents that can be administered with the albumin fusion protein and / or polynucleotide of the present invention include H ₂ histamine receptor antagonists (eg, TAGAMET ^™ (cimetidine), ZANTAC ^™ (ranitidine), PEPCID ^™ (famotidine), and AXID ^™ (nizatidine)); H ⁺ , K ⁺ ATPase (eg, PREVACID ^™ ) and PRILOSEC ^™ (omeprazole); Bismuth compounds (eg, PEPTO-BISMOL ^™ (bismuth subsalicylate) and DE-NOL (bismuth subcitrate) ^TM )); various antacids; sucralfate; prostaglandin analogs (eg CYTOTEC ^™ (misoprostol)); muscarinic choline Laxatives (eg, surface laxatives, irritant laxatives, saline and osmotic laxatives); antidiarrheal agents (eg, LOMOTIL ^™ (diphenoxylate), MOTOFEN ^™ (diphenoxin), and IMODIUM ^™ (roperamide hydrogen chloride)) , Synthetic analogs of somatostatin (eg, SANDOSTATIN ^™ (octreotide), antiemetics (eg, ZOFRAN ^™ (ondansetron), KYTRIL ^™ (granisetron hydrogen chloride), tropisetron, dolasetron, metoclopramide, chloropromazine, perphenazine, Procloperazine, Promethazine, Thiethylperazine, Triflupromazine, Domperidone, Haloperidol, Droperidol, Trimethobenzamide, Dexamethasone, Methi Prednisolone, dronabinol, and nabilone); D2 antagonists (metoclopramide, trimethobenzamide and chloropromazine); bile salts; chenodeoxycholine acid; ursodeoxycholine acid; and pancreatic enzyme preparations (eg, pancreas and pancreatic lipase) For example, but not limited to.

  In further embodiments, the albumin fusion proteins and / or polynucleotides of the invention are administered in combination with other therapeutic or prophylactic regimens (eg, radiation therapy).

  The present invention also provides a pharmaceutical pack or kit comprising one or more contents that are filled with one or more components of a pharmaceutical composition (including an albumin fusion protein of the present invention). Accompanying such a container may be a precautionary statement in the form directed to a government agency that controls the manufacture, use or sale of pharmaceutical and biological products. Reflects approval by the manufacturing, use or sales organization.

(Gene therapy)
The constructs encoding albumin fusion proteins of the invention can be used as part of a gene therapy protocol and can deliver a therapeutically effective dose of albumin fusion protein. A preferred approach for the in vivo introduction of nucleic acids into cells is by the use of viral vectors that contain nucleic acids encoding the albumin fusion proteins of the invention. The introduction of cells using viral vectors has the advantage that a large population of target cells can receive the nucleic acid.
In addition, molecules encoded within the viral vector (eg, as cDNA contained within the viral vector) are effectively expressed in the cell from which the viral vector nucleic acid was obtained.

  Retroviral and adenoviral vectors can be used as recombinant gene delivery systems for the transport of exogenous nucleic acid molecules that encode albumin fusion proteins in vivo. These vectors provide efficient nucleic acid delivery into the cell, and the transported nucleic acid is stably integrated into the host chromosomal DNA. The development of specialized cell lines (named “packaged cells”) that produce only replication-defective retroviruses increases the utility of retroviruses for gene therapy, and defective retroviruses for gene therapy purposes, (See, for example, Miller, (AD (1990) Blood 76: 271). Replication-deficient retroviruses are virions (use of helper viruses by standard techniques). Protocols for infecting cells with such viruses to generate recombinant retroviruses and in vivo or in vitro are described in Current Protocols in. Molecular Biolog , Ausubel, F.M., Et al. (Eds.) Greene Publishing Associates, (1989), may be found in 9.10-9.14 Section and other standard laboratory manuals.

  Another viral gene delivery system useful in the present invention uses adenovirus-derived vectors. The adenoviral genome can be processed so that it encodes and expresses the gene product of interest, but is inactivated in terms of its ability to replicate in the normal lytic viral life cycle. See, for example, Berkner et al., BioTechniques 6: 616 (1988); Rosenfeld et al., Science 252: 431-434 (1991); and Rosenfeld et al., Cell 68: 143-155 (1992). Suitable adenoviral vectors derived from the adenovirus strain Ad5 type d1324 or other adenovirus strains (eg, Ad2, Ad3, Ad7, etc.) are known to those skilled in the art. Recombinant adenoviruses can be advantageous in certain circumstances where they cannot infect non-dividing cells and infect a wide variety of cell types, including epithelial cells (Rosenfeld et al., (1992) cited above). In addition, viral particles are relatively stable and modifiable for purification and concentration, and can be modified to affect the infectious spectrum as described above. Viral DNA (and exogenous DNA contained therein) is not incorporated into the host cell genome, but remains in the episome, thereby inducing DNA to integrate into the host genome (eg, retrovirus). Avoid potential problems that can occur as a result of insertional mutagenesis, but large exogenous DNA (8 kilobases) Owned capacity of the adenoviral genome against, related to other gene delivery vectors (Berkner et al., Cited above; Haj-Ahmand et al., J.Virol.57: 267 (1986)).

  In another embodiment, the non-viral gene delivery of the invention relates to an endocytic pathway for absorption of a test nucleic acid molecule by a targeted cell. Exemplary gene delivery systems of this type include liposome-derived systems, polylysine conjugates, and artificial virus envelopes. In an exemplary embodiment, a nucleic acid molecule encoding an albumin fusion protein of the invention can be trapped in a liposome carrying a positive charge on its surface (eg, lipofectin) and (optionally) a target tissue Are tagged with antibodies (Mizuno et al. (1992) No Shinkei Geka 20: 547-5 5 1; PCT Publication WO 91/06309; Japanese Patent Application 1047381; and European Patent Publication EP-A -43075A).

  A gene delivery system for a gene encoding an albumin fusion protein of the present invention can be introduced into a patient by any number of methods. For example, pharmaceutical preparations of gene delivery systems can be introduced systemically (eg, by intravenous infusion) and specific conversion of proteins in specific target cells regulates the expression of gene delivery vehicles, receptor genes Provided by cell-type expression or tissue-type expression, or a combination thereof, due to the transcriptional regulatory sequences to be In other embodiments, the initial delivery of the recombinant gene is more limited by directing it to a reliably localized animal. For example, gene delivery vehicles can be induced by catheters (see US Pat. No. 5,328,470) or stereotaxic injection (eg, Chen et al. (1994) PNAS 91: 3054-3057). The pharmaceutical preparation of the gene therapy construct can consist essentially of the gene delivery system in an acceptable diluent or can include a delayed release matrix into which the gene delivery vehicle is incorporated. If the albumin fusion protein is produced intact from a recombinant cell (eg, a retroviral vector), the pharmaceutical preparation can include one or more cells that produce the albumin fusion protein.

(Further gene therapy method)
Also encompassed by the present invention are gene therapy methods for treating or preventing disorders, diseases and conditions. This gene therapy method involves the introduction of nucleic acid (DNA, RNA and antisense DNA or RNA) sequences into animals to achieve expression of the albumin fusion protein of the invention. This method requires a polynucleotide encoding an albumin fusion protein of the present invention operably linked to a promoter and any other genetic factors necessary for expression of the fusion protein by the target tissue. Such gene therapy and delivery techniques are known in the art (see, for example, WO 90/11092, incorporated herein by reference).

  Thus, for example, a patient-derived cell can be engineered with a polynucleotide (DNA or RNA) that includes a promoter operably linked to a polynucleotide encoding an albumin fusion protein of the invention ex vivo. The cells are provided to a patient to be treated with the fusion protein of the present invention. Such methods are well known in the art. For example, Belldegrun, A .; Et al. Natl. Cancer Inst. 85: 207-216 (1993); Ferrantini, M .; Cancer Research 53: 1107-1112 (1993); Ferrantini, M. et al. Et al. Immunology 153: 4604-4615 (1994); Kaido, T .; Et al., Int. J. et al. Cancer 60: 221-229 (1995); Ogura, H .; Cancer Research 50: 5102-5106 (1990); Santodonato, L. et al. Et al., Human Gene Therapy 7: 1-10 (1996); Santodonato, L. et al. Et al., Gene Therapy 4: 1246-1255 (1997); and Zhang, J. et al. -F. Et al., Cancer Gene Therapy 3: 31-38 (1996). These are incorporated herein by reference. In one embodiment, the engineered cell is an arterial cell. The arterial cells can be reintroduced to the patient via direct injection into the artery, tissue surrounding the artery, or via catheter injection.

  As discussed in more detail below, this polynucleotide construct can be used in any method of delivering injectable material to animal cells (eg, interstitial space in tissues (heart, muscle, skin, lung, liver, etc.)). Injection). The polynucleotide construct can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.

  In one embodiment, the polynucleotide encoding the albumin fusion protein of the invention is delivered as a naked polynucleotide. The term “naked” polynucleotide, DNA or RNA refers to any delivery vehicle (virus sequence, virus particle, liposome formulation, lipofectin or precipitate that acts to assist, facilitate or facilitate cell entry. (Including agents etc.). However, a polynucleotide encoding an albumin fusion protein of the invention can also be delivered, such as in liposome formulations and lipofectin formulations, which can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in US Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, which are hereby incorporated by reference. It is described in.

Polynucleotide vector constructs used in gene therapy methods are preferably constructs that do not integrate into the host genome or do not contain sequences that allow replication.
Suitable vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL available from Pharmacia; and pEF1 / V5, pcDNA3.1 and pRc available from Invitrogen. / CMV2. Other suitable vectors will be readily apparent to those skilled in the art.

  Any strong promoter known to those skilled in the art can be used to drive expression of the polynucleotide sequence. Suitable promoters include adenovirus promoters (eg, adenovirus major late promoter); or heterologous promoters (eg, cytomegalovirus (CMV) promoter); RS virus (RSV) promoter; inducible promoters (eg, MMT promoter, Metallothionein promoter); heat shock promoter; albumin promoter; ApoAI promoter; human globin promoter; viral thymidine kinase promoter (eg, herpes simplex thymidine kinase promoter); retroviral LTR; b-actin promoter; and human growth hormone promoter. This promoter may also be a native promoter for the polynucleotide of the present invention.

  Unlike other gene therapy techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transient nature of polynucleotide synthesis in those cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for a period of up to 6 months.

  Polynucleotide constructs can be used in tissues within animals (muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, intestine, testis, ovary, (Including the uterus, rectum, nervous system, eye, gland, and connective tissue). The interstitial space of this tissue is between the reticulofibers of the organ tissue, the intercellular fluid mucopolysaccharide matrix, the elastic fibers in the walls of the blood vessels or chambers, the collagen fibers of the fibrous tissue, or the connective tissue sheath muscle cells. including the same substrate in the tissue entraining muscle cell) or in the hiatus of the bone. This is likewise the space occupied by circulating plasma and lymph fluid in the lymph channels. Delivery to the interstitial space of muscle tissue is preferred for reasons discussed below. The polynucleotide constructs of the present invention can be conveniently delivered by injection into a tissue containing these cells. The polynucleotide constructs of the present invention are preferably delivered to and expressed in differentiated, persistent, non-dividing cells, but delivery and expression is non-differentiated cells or cells that are not fully differentiated ( For example, it can be achieved in blood stem cells or dermal fibroblasts). In vivo muscle cells are particularly qualified for their ability to take up and express polynucleotides.

  For naked nucleic acid sequence injections, an effective dosage of DNA or RNA is in the range of about 0.05 mg / kg body weight to about 50 mg / kg body weight. Preferably, the dosage is from about 0.005 mg / kg to about 20 mg / kg, and more preferably from about 0.05 mg / kg to about 5 mg / kg. Of course, as those skilled in the art will appreciate, this dosage will vary depending on the tissue site of the injection. Appropriate and effective dosages of the nucleic acid sequences can be readily determined by one skilled in the art and can depend on the condition being treated and the route of administration.

  The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes can also be used, including, for example, inhalation of aerosol formulations, particularly for delivery to lung or bronchial tissue, throat or nasal mucosa. Furthermore, the naked DNA construct can be delivered to the artery by the catheter used in this procedure during angioplasty.

  Naked polynucleotides are delivered by any method known in the art including, but not limited to, direct needle injection, intravenous injection, topical administration, catheter injection, and so-called “gene guns” at the delivery site. The These delivery methods are known in the art.

  This construct can also be delivered using delivery vehicles such as viral sequences, viral particles, liposomal formulations, lipofectin, precipitants, and the like. Such delivery methods are known in the art.

  In certain embodiments, the polynucleotide construct is complexed in a liposome preparation. Liposome preparations for use in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. However, cationic liposomes are particularly preferred because they can form a strong charge complex between the cationic liposome and the polyanionic nucleic acid. Cationic liposomes, in functional form, are plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. USA (1987) 84: 7413-7416), incorporated herein by reference. Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86: 6077-6081); and purified transcription factors (Debs, incorporated herein by reference). Et al., J. Biol. Chem. (1990) 265: 10189-10192) have been shown to mediate intracellular delivery.

  Cationic liposomes are readily available. For example, N [1-2,3-dioleyloxy) propyl] -N, N, N-triethylammonium (DOTMA) liposomes are particularly useful and are available under the trademark Lipofectin from GIBCO BRL, Grand Island, N .; Y. (See also Felgner et al., Proc. Natl. Acad. Sci. USA (1987) 84: 7413-7416, incorporated herein by reference). Other commercially available liposomes include transfectase (DDAB / DOPE) and DOTAP / DOPE (Boehringer).

  Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. For a description of the synthesis of DOTAP (1,2-bis (oleoyloxy) -3- (trimethylammonio) propane) liposomes, see for example PCT Publication No. WO 90/11092 (incorporated herein by reference). That. The preparation of DOTMA liposomes has been described in the literature and is described, for example, in P.D. Felgner et al., Proc. Natl. Acad. Sci. USA, 84: 7413-7417. Similar methods can be used to prepare liposomes from other cationic lipid substances.

  Similarly, anionic and neutral liposomes are readily available, for example, from Avanti Polar Lipids (Birmingham, Ala.) Or can be easily prepared using readily available materials. Such substances include phosphatidyl, choline, cholesterol, phosphatidylethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylethanolamine (DOPE), among others. These materials can also be mixed with DOTMA starting materials and DOTAP starting materials in appropriate proportions. Methods for producing liposomes using these materials are well known in the art.

  For example, commercially dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE) are used in various combinations, with or without the addition of cholesterol. Conventional liposomes can be produced. Thus, for example, DOPG / DOPC vesicles can be prepared by drying each 50 mg of DOPG and DOPC under a stream of nitrogen gas into a sonicated vial. The sample is placed under a vacuum pump overnight and hydrated with deionized water the next day. The sample was then placed in a capped vial using a Heat Systems model 350 sonicator equipped with an inverted cup (bath type) probe at the maximum setting while circulating the bath at 15EC. Sonicate for 2 hours. Alternatively, negatively charged vesicles can be prepared without sonication to produce multilamellar vesicles, or single sized membrane vesicles can be produced by extruding through a nuclear pore membrane. Can be generated. Other methods are known and available to those skilled in the art.

The liposome can include multilamellar vesicles (MLV), small unilamellar vesicles (SUV) or large unilamellar vesicles (LUV), with SUV being preferred. Various liposome-nucleic acid complexes are prepared using methods well known in the art. See, for example, Straubinger et al., Methods of Immunology (1983), 101: 512-527, incorporated herein by reference. For example, an MLV containing nucleic acid can be prepared by depositing a thin film of phospholipid on the wall of a glass tube and then hydrating with a solution of the substance to be encapsulated. SUVs are prepared by long-term sonication of MLVs to produce a homogeneous population of unilamellar liposomes. The substance to be encapsulated is added to the preformed suspension of MLV and then sonicated. When using liposomes containing cationic lipids, the dried lipid membrane is resuspended in a suitable solution, such as sterile water or an isotonic buffer solution such as 10 mM Tris / NaCl, sonicated, and then , Mix preformed liposomes directly with DNA. Due to the binding of positively charged liposomes to cationic DNA, liposomes and DNA form very stable complexes. SUVs find use with small nucleic acid fragments. LUVs are prepared by a number of methods well known in the art. Commonly used methods include Ca ²⁺ -EDTA chelation (Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394: 483; Wilson et al., Cell (1979)) 17:77); ether injection (Deamer, D. et al. And Bangham, A., Biochim.Biophys.Acta (1976) 443: 629; Ostro et al., Biochem.Biophys.Res. Communum. (1977) 76: 836; Fraley et al., Proc. Natl. Acad. Sci. ) 76: 3348); detergent dialysis (Enoch, H. and Strittmatter, P., Proc. Natl. Acad. Sci. USA (1979) 76: 145 And reverse phase evaporation (REV) (Fraley et al., J. Biol. Chem. (1980) 255: 10431; Szoka, F. and Papahadjopoulos, D., Proc. Natl. Acad. Sci. USA (1978) 75: 145; Schaefer-Ridder et al., Science (1982) 215: 166), which are hereby incorporated by reference.

  Generally, the ratio of DNA to liposome is from about 10: 1 to about 1:10. Preferably, the ratio is from about 5: 1 to about 1: 5. More preferably, the ratio is from about 3: 1 to about 1: 3. Even more preferably, the ratio is about 1: 1.

  US Pat. No. 5,676,954 (incorporated herein by reference) reports the injection of genetic material complexed with a cationic liposome carrier into mice. U.S. Pat. Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466, 5, 693,622, 5,580,859, 5,703,055 and International Publication No. WO 94/9469, which are hereby incorporated by reference, in which DNA is introduced into cells and Cationic lipids are provided for use in transfecting mammals. U.S. Pat. Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055 and International Publication No. WO 94/9469 (the present specification) (Incorporated herein by reference) provides a method of delivering a DNA-cationic lipid complex to a mammal.

  In certain embodiments, retroviral particles comprising RNA comprising a sequence encoding an albumin fusion protein of the invention are used to manipulate cells ex vivo or in vivo. Retroviruses from which retroviral plasmid vectors can be derived include Moloney murine leukemia virus, spleen necrosis virus, Rous sarcoma virus, Harvey sarcoma virus, chicken leukemia virus, gibbon leukemia virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and Examples include but are not limited to breast cancer virus.

This retroviral plasmid vector is used to transduce packaging cell lines to form producer cell lines. Examples of packaging cell lines that can be transfected include PE501, as described in Miller, Human Gene Therapy, 1: 5-14 (1990), which is incorporated herein by reference in its entirety. PA317, R-2, R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP + E-86, GP + envAm12 and DAN cell lines include, but are not limited to. This vector can transduce packaging cells by any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO ₄ precipitation. In one alternative, the retroviral plasmid vector can be encapsulated in liposomes or conjugated to lipids and then administered to the host.

  This producer cell line produces infectious retroviral vector particles comprising a polynucleotide encoding an albumin fusion protein of the invention. Such retroviral vector particles can then be used to transduce eukaryotic cells either in vitro or in vivo. This transduced eukaryotic cell expresses the fusion protein of the invention.

  In certain other embodiments, the polynucleotides contained in adenoviral vectors are used to manipulate cells ex vivo or in vivo. An adenovirus can be engineered such that it encodes and expresses the fusion protein of the invention and at the same time is inactivated with respect to its ability to replicate in the normal lytic viral life cycle. Adenovirus expression is achieved without integration of the viral DNA into the host cell chromosome, thus reducing concerns about insertional mutagenesis. In addition, adenoviruses have been used for many years as an intestinal live vaccine with excellent safety aspects (Schwartz, AR et al. (1974), Am. Rev. Respir. Dis., 109: 233-238). Finally, adenovirus-mediated gene transfer has been demonstrated in many examples including transfer of α-1-antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld, MA et al. (1991), Science. 252: 431-434; Rosenfeld et al. (1992) Cell 68: 143-155). Furthermore, extensive studies attempting to establish adenovirus as a causative agent in human cancer were uniformly negative (Green, M. et al. (1979) Proc. Natl. Acad. Sci. USA, 76: 6606. ).

  Suitable adenoviral vectors useful in the present invention are described, for example, in Kozarsky and Wilson, Curr. Opin. Genet. Dev. 3: 499-503 (1993); Rosenfeld et al., Cell 68: 143-155 (1992); Engelhardt et al., Human Genet. Ther. 4: 759-769 (1993); Yang et al., Nature Genet. 7: 362-369 (1994); Wilson et al., Nature 365: 691-692 (1993); and US Pat. No. 5,652,224, which are hereby incorporated by reference. Incorporated as. For example, the adenoviral vector Ad2 is useful and can be propagated in human 293 cells. These cells contain the E1 region of adenovirus and constitutively express Ela and Elb, which complement the defective adenovirus by providing the product of the gene that is missing from this vector. . In addition to Ad2, a variety of other adenoviruses (eg, Ad3, Ad5, and Ad7) are also useful in the present invention.

  Preferably, the adenovirus used in the present invention is replication defective. Replication defective adenoviruses require the help of helper virus and / or packaging cell lines to form infectious particles. The resulting virus is capable of infecting cells and can express the polynucleotide of interest operably linked to a promoter, but cannot replicate in most cells. Replication-deficient adenoviruses can be deleted in one or more of all or part of the following genes: E1a, E1b, E3, E4, E2a or L1-L5.

  In certain other embodiments, adeno-associated virus (AAV) is used to manipulate the cells ex vivo or in vivo. AAV is a naturally occurring defective virus that requires a helper virus to generate infectious particles (Muzyczka, N., Curr. Topics in Microbiol. Immunol. 158: 97 (1992)). AAV is also one of the few viruses that can incorporate its DNA into non-dividing cells. Vectors containing AAV as small as 300 base pairs can be packaged and integrated, but space for exogenous DNA is limited to about 4.5 kb. Methods for generating and using such AAV are known in the art. For example, U.S. Pat. Nos. 5,139,941, 5,173,414, 5,354,678, 5,436,146, 5,474,935, See 5,478,745 and 5,589,377.

  For example, suitable AAV vectors for use in the present invention contain all the sequences necessary for DNA replication, encapsidation, and host cell integration. Polynucleotide constructs are inserted into this AAV vector using standard cloning methods, such as those found in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989). The recombinant AAV vector is then transfected into packaging cells infected with helper virus using any standard technique, including lipofection, electroporation, calcium phosphate precipitation, and the like. Suitable helper viruses include adenovirus, cytomegalovirus, vaccinia virus, or herpes virus. Once the packaging cells are transfected and infected, they produce infectious AAV virions that contain the polynucleotide construct. These viral particles are then used to transduce eukaryotic cells, either ex vivo or in vivo. This transduced cell contains the polynucleotide construct integrated into its genome and expresses the fusion protein of the invention.

  Another method of gene therapy is homologous recombination (eg, US Pat. No. 5,641,670, issued June 24, 1997; International Publication No. WO 96/29411, published September 26, 1996; International Publication). WO 94/12650, published Aug. 4, 1994; see Koller et al., Proc. Natl. Acad. Sci. USA 86: 8932-8935 (1989); And operably linking the heterologous regulatory region and the endogenous polynucleotide sequence (eg, a sequence encoding a polypeptide of the present invention). This method involves the activation of a gene that is present in the target cell but is usually not expressed in that cell or expressed at a lower level than desired.

  Polynucleotide constructs are made using standard techniques known in the art. This construct includes a promoter with a targeting sequence adjacent to the promoter. Suitable promoters are described herein. The targeting sequence is sufficiently complementary to the endogenous sequence to allow homologous recombination between the promoter-targeting sequence and the endogenous sequence. The targeting sequence is present sufficiently close to the 5 'end of the desired endogenous polynucleotide sequence, and thus upon homologous recombination, the promoter is operably linked to the endogenous sequence.

  This promoter and targeting sequence can be amplified using PCR. Preferably, the amplified promoter contains separate restriction enzyme sites at the 5 'and 3' ends. Preferably, the 3 ′ end of the first targeting sequence contains the same restriction enzyme sites as the 5 ′ end of the amplified promoter and the 5 ′ end of the second targeting sequence is 3 of the amplified promoter. 'Contains the same restriction sites as the ends. The amplified promoter and targeting sequence are digested and ligated together.

  Either as a naked polynucleotide or with transfection facilitating agents such as liposomes, viral sequences, viral particles, whole viruses, lipofections, precipitants etc. as described in more detail above. A promoter-targeting sequence construct is delivered to the cell. The P promoter-targeting sequence can be delivered by any method including direct needle injection, intravenous injection, local administration, catheter injection, particle accelerator, and the like. This method is described in more detail below.

  The promoter-targeting sequence construct is taken up by the cell. Homologous recombination occurs between this construct and the endogenous sequence, so that the endogenous sequence is placed under the control of this promoter. This promoter then drives the expression of the endogenous sequence.

  The polynucleotide encoding the albumin fusion protein of the present invention contains a secretory signal sequence that facilitates secretion of the protein. Typically, this signal sequence is located in the coding region of the polynucleotide which is expressed towards or at the 5 'end of the coding region. This signal sequence may be homologous or heterologous to the polynucleotide of interest, and may be homologous or heterologous to the cell to be transfected. . In addition, the signal sequence can be chemically synthesized using methods known in the art.

  Any dosage form of any of the above polynucleotide constructs can be used as long as the dosage form expresses one or more molecules in an amount sufficient to provide a therapeutic effect. This includes direct needle injection, systemic injection, catheter injection, biolistic syringes, particle accelerators (ie, “gene guns”), gelfoam sponge depots, other commercial depot materials, osmotic agents Includes pressure pumps (eg, Alza minipumps), solid (tablets or pills) pharmaceutical formulations for oral or suppository, and decanting or topical application during surgery. For example, direct injection of calcium phosphate precipitated naked plasmid into rat liver and rat spleen, or direct injection of protein-coated plasmid into portal vein resulted in gene expression of foreign genes in rat liver (Kaneda et al., Science, 243 : 375 (1989)).

  The preferred method of local administration is by direct injection. Preferably, the albumin fusion protein of the present invention complexed with a delivery vehicle is administered by direct injection within the arterial region or locally administered within the arterial region. Topical administration of a composition within the arterial area refers to injecting the composition into the artery several centimeters, preferably several millimeters.

  Another method of topical administration is to contact the polynucleotide construct of the present invention in or around a surgical wound. For example, the patient can experience surgery and the polynucleotide construct can be coated on the surface of the tissue inside the wound, or the construct can be injected into a region of tissue inside the wound.

  A therapeutic composition useful for systemic administration comprises a fusion protein of the invention complexed with a targeted delivery vehicle of the invention. Suitable delivery vehicles for use in systemic administration include liposomes containing a ligand that targets the vehicle to a specific site. In certain embodiments, suitable delivery vehicles for use in systemic administration include liposomes comprising an albumin fusion protein of the invention for targeting the vehicle to a specific site.

Preferred methods of systemic administration include intravenous injection, aerosol, oral and transdermal (topical) delivery. Intravenous injection can be performed using standard methods in the art.
Aerosol delivery can also be performed using standard methods in the art (eg, Stribling et al., Proc. Natl. Acad. Sci. USA 189: 11277-11281, incorporated herein by reference. (See 1992). Oral delivery can be performed by complexing the polynucleotide construct of the present invention to a carrier capable of withstanding degradation by digestive enzymes in the intestine of the animal. Examples of such carriers include plastic capsules or tablets, such as those known in the art. Topical delivery can be performed by mixing a lipophilic reagent that can pass into the skin (eg, DMSO) and a polynucleotide construct of the invention.

  Determining an effective amount of a substance to be delivered includes, for example, the chemical structure and biological activity of the substance, the age and weight of the animal, the exact condition requiring treatment and its severity, and the route of administration Can depend on a number of factors. The frequency of treatment depends on many factors, such as the amount of polynucleotide construct administered per dose and the health and medical history of the subject. The exact amount, number of doses and timing of dosing will be determined by the attending physician or attending veterinarian.

  The albumin fusion protein of the present invention can be administered to any animal, preferably to mammals and birds. Preferred mammals include humans, dogs, cats, mice, rats, rabbits, sheep, cows, horses and pigs, with humans being particularly preferred.

(Biological activity)
The albumin fusion proteins and / or polynucleotides encoding albumin fusion proteins of the invention can be used in assays that test one or more biological activities. When an albumin fusion protein and / or a polynucleotide encoding an albumin fusion protein show activity in a particular assay, the therapeutic protein corresponding to the fusion protein is involved in a disease associated with its biological activity. There is a possibility. Thus, the fusion protein can be used for the treatment of related diseases.

  In a preferred embodiment, the present invention includes the steps of treating the diseases and disorders listed in the “Preferred Indicator Y” column of Table 1, administering to the patient, wherein such treatment, prevention, Alternatively, an improvement would desire an albumin fusion protein of the invention that includes a therapeutic protein portion that matches the therapeutic protein, which therapeutic protein is listed in the “Therapeutic Protein X” (Table 1 “Preferred Indicator Y” column). As fresh as the disease or disorder to be treated.

  In a further preferred embodiment, the invention encompasses a method of treating a disease or disorder listed for a particular therapeutic protein in the “preferred indicator: Y” column of Table 1, comprising the step of administering to a patient, Here, such treatment, prevention or amelioration is desired for albumin fusion proteins of the present invention comprising a therapeutic protein portion consistent with the therapeutic protein, wherein the indicators in the examples are effective for treatment, prevention or amelioration Related in a quantity.

  When encoded by the polynucleotide encoding SEQ ID NO: Y, it is specifically contemplated by the present invention that an albumin fusion protein is produced from the cell. When a polynucleotide is used to express a cell-derived encoded protein, the cell's natural secretion and processing steps produce a protein that lacks the signal sequence clearly listed in columns 4 and / or 11 of Table 2. To do. The specific amino acid sequences of the listed signal sequences are set forth herein or are well known in the art. Thus, the most preferred embodiment of the present invention comprises an albumin fusion protein derived from cells (which lacks the leader sequence as shown in columns 4 and / or 11 of Table 2). The most preferred poliopeptide comprises SEQ ID NO: Y without the specific leader sequence listed in columns 4 and / or 11 of Table 2. Also preferred are compositions (including pharmaceutical compositions) comprising these two preferred embodiments. These albumin fusion proteins are specifically contemplated for the treatment, prevention, or amelioration of the diseases or disorders listed in Table 1 “Preferred Indicator: Y Column for a particular therapeutic protein.

  In preferred embodiments, the fusion proteins of the invention comprise endocrine diseases and disorders (see, eg, “endocrine disorders” in the following section), nervous system diseases and disorders (see, eg, “neurology” in the following section). ), Diseases and disorders of the immune system (eg see “Immunoactivity” in the following section), diseases and disorders of the respiratory system (eg “respiratory organs in the following section) Disorders ”), cardiovascular diseases and disorders (eg, see“ Cardiovascular disorders ”in the following section), reproductive diseases and disorders (eg,“ reproductive disorders ”in the following section) ), Digestive system diseases and disorders (see, eg, “Gastrointestinal Disorders” in the following section), diseases and / or disorders associated with cell proliferation (eg, , See “Hyperproliferative disorders” in the following section), and / or diseases associated with blood-related diseases and / or disorders (see, for example, “blood-related disorders” in the following section) and It may be used in the diagnosis, prognosis, prevention and / or treatment of disorders.

  In certain embodiments, the albumin fusion proteins of the invention are used for diagnosis and / or prognosis of diseases and / or disorders associated with tissues in which genes corresponding to some of the therapeutic proteins of the fusion proteins of the invention are expressed. sell.

  Therefore, the fusion protein of the present invention and the polynucleotide encoding the albumin fusion protein of the present invention are useful for diagnosis, detection and / or treatment of diseases and / or disorders associated with the following activities, and as these activities: Include, but are not limited to, hormone activation, neurotransmission activity, cell signaling, cell proliferation, cell differentiation, and cell migration.

  More generally, the fusion proteins of the present invention and polynucleotides encoding the albumin fusion proteins of the present invention can be used for the diagnosis, prognosis, prevention and / or treatment of diseases and / or disorders associated with the following systems.

(Immunoactivity)
The albumin fusion protein of the present invention and the polynucleotide encoding the albumin fusion protein of the present invention are, for example, immune system diseases and disorders caused by activating or inhibiting the proliferation, differentiation or mobilization (chemotaxis) of immune cells. And / or the condition may be useful in treating, preventing, diagnosing and / or prognosing. Immune cells arise through a process called hematopoiesis and produce myeloid cells (platelets, red blood cells, neutrophils and macrophages) and lymphoid cells (B lymphocytes and T lymphocytes) from pluripotent stem cells. The etiology of these immune diseases, disorders and / or conditions can be genetic, somatic (eg, cancer and some autoimmune disorders), acquired (eg, due to chemotherapy or toxins) or infectious It can be. Furthermore, the fusion proteins of the invention and the polynucleotides encoding the albumin fusion proteins of the invention can be used as markers or detectors for diseases or disorders of a particular immune system.

  In another embodiment, the fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used to treat diseases and disorders of the immune system and / or in which the polypeptide of the invention is expressed. Can be used to inhibit or enhance the immune response generated by cells associated with.

  The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can be used for the treatment, prevention, diagnosis and / or prognosis of immunodeficiencies (including both congenital or acquired immunodeficiencies). Can be useful to. Examples of B cell immunodeficiency in which immunoglobulin levels, B cell function and / or number of B cells are reduced include the following: X-linked agammaglobulinemia (Bruton's disease), X-linked pediatric agamma Globulinemia, X-linked immunodeficiency with excess IgM, non-X-linked immunodeficiency with excess IgM, X-linked lymphoproliferative syndrome (XLP), agammaglobulinemia (congenital and acquired agammaglobulinemia) Adult-onset agammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia, unspecified hypogammaglobulinemia, recessive agammaglobulinemia (Swiss type), selective IgM deficiency, selective IgA deficiency, selective IgG subclass deficiency, IgG subclass Deficiency (with or without IgA deficiency), Ig deficiency (with increased IgM), IgG and IgA deficiency (with increased IgM), antibody deficiency with normal Ig or elevated Ig Ig heavy chain deficiency, kappa chain deficiency, B cell lymphoproliferative disorder (BLPD), non-classifiable immunodeficiency (CVID), non-classifiable immunodeficiency (CVI) (acquired), and transient infant low gamma Globulinemia.

  In certain embodiments, telangiectasia ataxia or a condition associated with telangiectasia ataxia is treated using a fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention. Prevention, diagnosis and / or prognosis.

  Examples of congenital immunodeficiencies in which the function and / or number of T cells and / or B cells are reduced include, but are not limited to: Di George abnormality, severe combined immunodeficiency ( SCID) (X-linked SCID, autosomal recessive SCID, adenosine deaminase deficiency, purine nucleoside phosphorylase (PNP) deficiency, class II MHC deficiency (deficient lymphocyte syndrome), Viscott-Oldrich syndrome, and telangiectasia Including but not limited to ataxia), thymic dysgenesis, 3rd and 4th pharyngeal sac syndrome, 22q11.2 deficiency, chronic mucocutaneous candidiasis, natural killer cell deficiency (NK), idiopathic CD4 + T lymphocytes Reduction, immunodeficiency with dominant T cell deficiency (unspecified), and cell-mediated immunity Specific immunodeficiency.

  In certain embodiments, a Di George abnormality or a condition associated with a Di George abnormality is treated, prevented, diagnosed using a fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention. And / or prognosis.

  Other immunodeficiencies that can be treated, prevented, diagnosed and / or prognosticated using the fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention include the following: Not limited to: Chronic granulomatosis, Chediac-East disease, myeloperoxidase deficiency, lymphocyte glucose-6-phosphate dehydrogenase deficiency, X-linked lymphoproliferative syndrome (XLP), lymphocyte adhesion deficiency, complement Body component deficiency (including C1, C2, C3, C4, C5, C6, C7, C8 and / or C9 deficiency), reticular dysgenesis, thymic lymphogenesis, dysgenesis with thymoma, severe Coexistence of congenital lymphopenia, dysplasia with immunodeficiency, neonatal neutropenia, dwarfism and Ig deficiency That Nezerofu syndrome.

  In a preferred embodiment, these immunodeficiencies and / or conditions associated with the above-mentioned immunodeficiencies are treated, prevented using the fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention. Can be diagnosed and / or prognosticated.

  In a preferred embodiment, the fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention can be used as an agent to boost immune responsiveness among immunocompromised individuals. In certain embodiments, a fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention is used as an agent to boost immune responsiveness among B cell and / or T cell immunodeficient individuals. Can be done.

  The albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention may be useful in the treatment, prevention, diagnosis and / or prognosis of autoimmune disorders. Many autoimmune disorders are caused by inappropriate recognition of self substances as foreign substances by immune cells. This inappropriate recognition results in an immune response that induces destruction of host tissue. Accordingly, administration of a fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention capable of inhibiting an immune response, particularly T cell proliferation, differentiation or chemotaxis, prevents autoimmune disorders. Can be an effective treatment.

  Examples of the autoimmune disease or disorder that can be treated, prevented, diagnosed and / or prognosticated by the fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention include one or more of the following: Not limited to: systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, multiple sclerosis, autoimmune thyroiditis, Hashimoto's thyroid disease, autoimmune hemolytic anemia, hemolytic anemia, thrombocytopenia, autoimmune thrombocytopenia Purpura, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia purpura, purpura (eg, Henojo-Schönlein purpura), autoimmune cytopenias, Goodpasture syndrome, pemphigus vulgaris, severe muscle Asthenia, Graves' disease (hyperthyroidism), and insulin-resistant diabetes.

  Additional disorders that may have an autoimmune component that can be treated, prevented and / or diagnosed with an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention include, but are not limited to: Not limited to: Type II collagen-induced arthritis, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, neuritis, uveitis ophthalmitis, multiple Endocrinopathy, Reiter's disease, stiff man syndrome, autoimmune pneumonia, autism, Giant-Barre syndrome, insulin-dependent diabetes mellitus, and autoimmune inflammatory eye disorders.

  Additional disorders that may have an autoimmune component that can be treated, prevented, diagnosed and / or prognosticated with an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention include the following: But are not limited to: scleroderma with anti-collagen antibodies (eg often characterized by nucleolus and other nuclear antibodies), mixed connective tissue disease (eg soluble nuclear antigens (eg ribonucleoprotein)) Often characterized by antibodies against), polymyositis (eg often characterized by non-histone ANA), pernicious anemia (eg often characterized by antimural cells, microsomes, and intrinsic factor antibodies), idiopathic addison Disease (eg, humoral and cell-mediated adrenal glands) Often characterized by alveolar toxicity), infertility (eg often characterized by anti-sperm antibodies), glomerulonephritis (eg often characterized by glomerular basement membrane antibodies or immune complexes), bullous species Pemphigus (eg, often characterized by IgG and complement in the basement membrane), Sjogren's syndrome (eg, often characterized by multi-tissue antibodies and / or certain non-histone ANA (SS-B)), diabetes ( For example, often characterized by cell-mediated and humoral islet cell antibodies), and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis) (eg, β-adrenergic receptors) Often characterized by antibodies).

  Additional disorders that may have an autoimmune component that can be treated, prevented, diagnosed and / or prognosticated with an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention include the following: But are not limited to: chronic active hepatitis (eg, often characterized by smooth muscle antibodies), primary biliary cirrhosis (eg, often characterized by mitochondrial antibodies), other endocrine insufficiency (eg, some Often characterized by specific tissue antibodies in such cases), vitiligo (eg often characterized by melanocyte antibodies), vasculitis (eg often Ig and complement in the vessel wall and / or low serum complement) Characterized), after MI For example, often characterized by myocardial antibodies), open heart syndrome (eg often characterized by myocardial antibodies), urticaria (eg often characterized by IgG and IgM antibodies to IgE), atopic dermatitis ( Often characterized by IgG and IgM antibodies to IgE), asthma (eg, often characterized by IgG and IgM antibodies to IgE), and many other inflammatory, granulomatous, degenerative and atrophic disorders.

  In preferred embodiments, these autoimmune diseases and disorders and / or disorders and / or conditions associated with these diseases and disorders described above encode, for example, a fusion protein of the invention and / or an albumin fusion protein of the invention. Polynucleotides that are treated, prevented, diagnosed and / or prognosticated. In certain preferred embodiments, rheumatoid arthritis is treated, prevented and / or diagnosed using a fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention.

  In another particular preferred embodiment, systemic lupus erythematosus is treated, prevented, diagnosed and / or predicted using a fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention. In another particular preferred embodiment, idiopathic thrombocytopenic purpura is treated, prevented and / or diagnosed using the fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention.

  In another particular preferred embodiment, IgA nephropathy is treated, prevented and / or diagnosed using a fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention.

  In preferred embodiments, autoimmune diseases and autoimmune disorders and / or conditions associated with the aforementioned diseases and disorders are treated with the fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention. Prevention, diagnosis and / or prediction.

  In a preferred embodiment, the fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used as an immunosuppressant.

  The albumin fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention may be useful in the treatment, prevention, prediction and / or diagnosis of hematopoietic cell diseases, disorders and / or conditions. The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention includes specific, but not limited to, leukopenia, neutropenia, anemia, and thrombocytopenia It can be used to increase the differentiation and proliferation of hematopoietic cells, including pluripotent stem cells, in an attempt to treat or prevent diseases, disorders and / or conditions associated with the reduction of (or many) types of hematopoietic cells. Alternatively, the fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention may be a reduction of a particular (or many) type of hematopoietic cells, including but not limited to histiocytosis. Can be used to increase the differentiation and proliferation of hematopoietic cells, including pluripotent stem cells, in an attempt to treat or prevent diseases, disorders and / or conditions associated with.

  Allergic reactions and conditions (eg, asthma (especially allergic asthma) or other respiratory disorders) can also be treated, prevented, diagnosed and / or predicted using a polynucleotide encoding a fusion protein of the invention. In addition, these molecules can be used to treat, prevent, predict and / or diagnose anaphylaxis, hypersensitivity to antigenic molecules, or blood group incompatibility.

Furthermore, a polynucleotide encoding a fusion protein of the invention can be used to treat, prevent, diagnose and / or predict an IgE-mediated allergic response. Such allergic reactions include, but are not limited to, asthma, rhinitis and eczema.
In certain embodiments, a polynucleotide encoding a fusion protein of the invention may be useful for modulating IgE concentration in vitro or in vivo.

  Furthermore, the polynucleotide encoding the fusion protein of the present invention is used for diagnosis, prediction, prevention and / or treatment of inflammatory conditions. For example, polynucleotides encoding the fusion proteins of the invention can inhibit the activation, proliferation and / or differentiation of cells involved in inflammatory responses, so these molecules can be used for both chronic and acute conditions. The inflammatory condition of can be prevented and treated. Such inflammatory conditions include, but are not limited to: For example, inflammation associated with infection (eg, septic shock, sepsis, or systemic inflammatory response syndrome), inflammation associated with ischemia-reperfusion injury, inflammation associated with endotoxin lethality, complement mediated hyperacute rejection Inflammation, inflammation associated with nephritis, inflammation associated with cytokines or chemokine-induced lung injury, inflammation associated with inflammatory bowel disease, inflammation associated with Crohn's disease or excess of cytokines (eg, TNF or IL-1) Inflammation resulting from generation, respiratory disorders (eg, asthma and allergies); gastrointestinal disorders (eg, inflammatory bowel disease); cancers (eg, gastric cancer, ovarian cancer, lung cancer, bladder cancer, liver cancer, and breast cancer); CNS disorders (Eg, multiple sclerosis, ischemic brain injury and / or stroke, traumatic brain injury, neurodegenerative disorders (eg, Parkinson's disease and Alzheim -Disease), AIDS-related dementia, and prion disease); cardiovascular disorders (eg, atherosclerosis, myocarditis, cardiovascular disease, and cardiopulmonary bypass complications); and many additional diseases characterized by inflammation, Conditions and disorders (eg, hepatitis, rheumatoid arthritis, gout, trauma, pancreatitis, sarcoidosis, dermatitis, renal ischemia reperfusion injury, Graves' disease, systemic lupus erythematosus, diabetes mellitus, and allograft rejection).

  Because inflammation is a basic defense mechanism, inflammatory disorders can affect virtually any tissue in the body. Accordingly, the polynucleotides encoding the fusion proteins of the invention are used for the treatment of tissue specific inflammatory disorders, including but not limited to the following. Adrenalitis, alveolitis, cholangitis, appendicitis, balditis, blepharitis, bronchitis, synovial inflammation, carditis, cellulitis, uterine tractitis, cholecystitis, vocal cord inflammation, cochulitis, colitis, conjunctivitis, Cystitis, dermatitis, diverticulitis, encephalitis, endocarditis, esophagitis, otitis, fibrohistitis, follicular inflammation, gastritis, gastroenteritis, gingivitis, glossitis, hepatosplenitis, keratitis, labyrinthitis , Laryngitis, lymphangitis, mastitis, mediastinitis, meningitis, uteritis, mucositis, myocarditis, myositis, tympanitis, nephritis, neuritis, testitis, osteomyelitis, otitis, pericarditis, Peritonitis, peritonitis, laryngitis, phlebitis, gray pulpitis, prostatitis, pulpitis, retinitis, rhinitis, fallopianitis, scleritis, scleral choroiditis, scrotitis, sinusitis, spondylitis, Steatitis, stomatitis, synovitis, otitis, tendonitis, tonsillitis, urethritis, and vaginitis.

In certain embodiments, a polynucleotide encoding a fusion protein of the invention is useful for diagnosis, prediction, prevention and / or treatment of organ transplant rejection and graft versus host disease. Organ rejection occurs by host immune cell destruction of transplanted tissue through an immune response.
Similarly, the immune response is also related to GVHD, where exogenous transplanted immune cells destroy host tissue. Polypeptides, antibodies, or polynucleotides of the invention and / or agonists or antagonists thereof (inhibiting immune responses, particularly T cell activation, proliferation, differentiation, or chemotaxis) prevent organ rejection or GVHD It can be an effective treatment. In certain embodiments, a polynucleotide encoding a fusion protein of the invention (which inhibits immune responses, particularly T cell activation, proliferation, differentiation, or chemotaxis) is an experimental allergy rejection and hyperacute xenograft It can be an effective treatment to prevent rejection.

  In other embodiments, the polynucleotide encoding the fusion protein of the invention may be useful in the diagnosis, prediction, prevention and / or treatment of immune complex diseases, including but not limited to the following. Serum disease, post streptococcal glomerulonephritis, polyarteritis nodosa, and immune complex-induced vasculitis.

  A polynucleotide encoding an albumin fusion protein of the invention can be used for the treatment, detection and / or prevention of infectious agents. For example, infectious diseases can be treated, detected, and / or prevented by increasing the immune response, in particular by increasing the proliferation, activation and / or differentiation of B cells and / or T cells. The immune response can be increased by either increasing an existing immune response or eliciting a new immune response. Alternatively, a polynucleotide encoding a fusion protein of the invention can also directly inhibit infectious agents (as described in the section on application of infectious agents, etc.) without the inevitable triggering of an immune response.

  In another embodiment, a polynucleotide encoding an albumin fusion protein of the invention is used as a vaccine adjuvant that increases the immune response to the antigen. In certain embodiments, a polynucleotide encoding an albumin fusion protein of the invention is used as an adjuvant to increase a tumor-specific immune response.

  In another specific embodiment, the polynucleotide encoding the fusion protein of the invention is used as an adjuvant to increase the antiviral immune response. Antiviral immune responses that can be enhanced using the compositions of the present invention as adjuvants include viruses and virus-related diseases or conditions described herein or otherwise known in the art. In certain embodiments, the composition of the invention comprises an immune response against a virus, disease or condition selected from the group consisting of: AIDS, meningitis, dengue, EBV, and hepatitis (eg, hepatitis B). Used as an adjuvant to enhance In another specific embodiment, the composition of the invention comprises the following: HIV / AIDS, RS virus, dengue, rotavirus, Japanese encephalitis, influenza A and B, parainfluenza, measles, cytomegalovirus, rabies, Junin virus Used as an adjuvant to enhance the immune response to a virus, disease or condition selected from the group consisting of Chikungunya virus, Rift Valley fever, herpes simplex, and yellow fever.

  In another specific embodiment, a polynucleotide encoding an albumin fusion protein of the invention is used as an adjuvant to increase an antibacterial or antifungal immune response. Anti-bacterial or anti-fungal immune responses that can be augmented using the compositions of the present invention as an adjuvant include bacteria or fungi, and are described herein or otherwise known in the art Bacteria or fungi associated with other diseases or conditions. In certain embodiments, the compositions of the present invention increase the immune response against bacteria or fungi, diseases, or symptoms, which are selected from the group consisting of tetanus, diphtheria, botulism, and type B meningitis. To be used as an adjuvant.

  In another specific embodiment, the composition of the present invention comprises a bacterium or fungus, disease, or symptom (including Vibrio cholerae, Mycobacterium leprae, Salmonella typhi, Salmonella parasityphi, Meisseria meningitido, Spp., Enterotoxigenic Escherichia coli, Enterohemorrhagic E. coli, and Borrelia burgdorferi are used as an adjuvant to increase the immune response.

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used as an adjuvant to increase the antiparasitic immune response. Antiparasitic immune responses that can be augmented using the compositions of the invention as adjuvants include those associated with parasites and diseases or conditions described or otherwise known in the art. And parasites that In certain embodiments, the compositions of the invention are used as adjuvants to increase the immune response against parasites. In another specific embodiment, the compositions of the invention are used as an adjuvant to increase the immune response against Plasmodium (malaria) or Leishmania.

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention can also be treated by, for example, preventing the recruitment and activation of mononuclear phagocytes. It can be used to treat infectious diseases including embryosis, psychoidosis, and idiopathic pulmonary fibrosis.

  In another specific embodiment, an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention produces an antibody that inhibits or increases an immune-mediated response to the polypeptide of the invention. Used as an antibody for.

  In one embodiment, an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention boosts the immune system and provides one or more antibodies (eg, IgG, IgA, IgM, and IgE). ) In order to induce high affinity antibody production and immunoglobulin class switching (eg, IgG, IgA, IgM and IgE) and / or increase immune response (eg, mouse, Rats, rabbits, hamsters, guinea pigs, pigs, minipigs, chickens, camels, goats, horses, cows, sheep, dogs, cats, non-human primates, and humans, most preferably humans).

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used as a B cell responsive stimulator against a pathogen.

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used as an activator of T cells.

  In another specific embodiment, an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention is used as an agent to increase an individual's immune status prior to receiving immunosuppressive treatment. The

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used as an agent to induce higher affinity antibodies.

  In another specific embodiment, an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention is used as an agent to increase serum immunoglobulin levels.

  In another specific embodiment, an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention is used as an agent to promote recovery of an immunocompromised individual.

  In another specific embodiment, an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention is used as an agent to boost immune responsiveness between an elderly population and a newborn. The

  In another specific embodiment, an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention can be used for bone marrow transplantation and / or other transplantation (eg, allogeneic or exogenous organ transplantation). ) Used as an immune system enhancer before, during or after. With respect to transplantation, the compositions of the invention can be administered before, contemporaneously and / or after transplantation. In certain embodiments, the compositions of the invention are administered after transplantation and before initiation of T cell population recovery. In another specific embodiment, the composition of the invention is administered first after transplantation, after the onset of T cell population recovery, but prior to full recovery of the B cell population.

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is for boosting immune responsiveness among individuals with an acquired defect in B cell function. Used as a medicine. Conditions resulting in an acquired defect of B cell function that can be ameliorated or treated by administering an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention include HIV infection, AIDS, bone marrow Examples include, but are not limited to, transplantation and B-cell chronic lymphocytic leukemia (CLL).

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used as an agent for boosting immune responsiveness among individuals with transient immune deficiency used. Conditions that result in transient immunodeficiency that can be ameliorated or treated by administering an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention include from viral infections (eg, influenza). Including, but not limited to, recovery from malnutrition, conditions associated with malnutrition, recovery from infectious mononucleosis, or conditions related to stress, recovery from measles, recovery from blood transfusions, and recovery from surgery .

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used as a regulator of antigen presentation by monocytes, dendritic cells and / or B cells. . In one embodiment, an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention enhances or antagonizes antigen presentation in vitro or in vivo. Further, in related embodiments, this enhanced antigen presentation or antagonization may be useful as an anti-tumor treatment or to modulate the immune system.

  In another specific embodiment, an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention can cause an individual's immune system to undergo a humoral response (i.e., as opposed to a THl cellular response). , TH2) is used as a drug for directing the generation of TH2.

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention induces tumor growth and thus makes the tumor more sensitive to anti-tumor agents. Used as a means to do. For example, multiple myeloma is a disease of slow cell division and is therefore refractory to virtually all anti-tumor regimens. If these cells are grown more rapidly, their sensitivity profile will likely change.

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention comprises AIDS, chronic lymphocyte disorder and / or non-classifiable immunodeficiency. As a stimulator of B cell production in pathologies such as

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is for the generation and / or regeneration of lymphoid tissue after surgery, trauma or genetic defect Used as a treatment. In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used as a pretreatment of a bone marrow sample prior to transplantation.

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is heritable to produce an immunodeficiency / immune deficiency as observed among SCID patients. Used as a gene-based treatment for disorders.

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used in parasite diseases that affect monocytes (eg, Leishmania). Used as a means of activating monocytes / macrophages to protect against.

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used as a means of modulating secreted cytokines elicited by the polypeptide of the invention.

  In another embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention can be applied to one or more of the herein described, as can be applied to veterinary medicine. Used in application.

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used as a means to block various aspects of the immune response against foreign factors or self. Examples of diseases or conditions that block the immune response of certain aspects that may be desired include autoimmune disorders such as lupus and arthritis, and skin allergies, inflammation, bowel disease, immune responsiveness to injury and pathogen related diseases / disorders Is mentioned.

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is an autoimmune disease (eg, idiopathic thrombocytopenic purpura, systemic lupus erythematosus and MS). Used as a treatment to prevent B cell proliferation and Ig secretion associated with

  In another specific embodiment, the polypeptide, antibody, polynucleotide of the invention and / or the agonist or antagonist of the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is an endothelial cell. As an inhibitor of B cell and / or T cell migration. This activity disrupts tissue structures or cognate responses and is useful, for example, in disrupting immune responses and blocking sepsis.

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is non-quantitative monoclonal gammapathy of undetermined signature. ) (MGUS), used as a treatment for chronic hypergammaglobulinemia events in diseases such as Waldenstrom disease, idiopathic monoclonal gammaglobulinemia, and plasmacytoma. The

  In another specific embodiment, the composition of the invention comprises, for example, macrophages and their precursors, and neutrophils, basophils, B lymphocytes in certain autoimmune and chronic inflammatory and infectious diseases. And several T cell subsets such as activated T cells and CD8 cytotoxic T cells and natural killer cells can be used to inhibit polypeptide chemotaxis and activation. Examples of autoimmune diseases are described herein and examples of autoimmune diseases include multiple sclerosis and insulin-dependent diabetes.

  An albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention may also be used to treat idiopathic eosinophilia syndrome, for example, by preventing eosinophil production and migration. Can be used.

  In another specific embodiment, an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention is used to increase or inhibit complementary intervening cell lysis.

  In another specific embodiment, an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention is used to increase or inhibit antibody-dependent cellular cytotoxicity.

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention may also reduce atherosclerosis, for example by preventing monocyte infiltration in the arterial wall. Can be used to treat.

  In another specific embodiment, an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention can be used to treat adult respiratory distress syndrome (ARDS).

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention may stimulate stimulation of wound and tissue repair, stimulation of angiogenesis, and / or blood vessels or It may be useful in stimulating repair of lymphatic diseases or disorders. Furthermore, the albumin fusion protein of the invention and / or the polynucleotide encoding the fusion protein of the invention can be used to stimulate mucosal surface regeneration.

  In certain embodiments, an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention comprises primary or acquired immune deficiency, defective serum immunoglobulin production, recurrent infection and Used to diagnose, predict, treat and / or prevent disorders characterized by immune system dysfunction. Further, the albumin fusion protein of the present invention and / or the polynucleotide encoding the fusion protein of the present invention may be used for infection of joints, bones, skin and / or parotid gland, blood-derived infection (eg, sepsis, meningitis, Septic arthritis and / or osteomyelitis), autoimmune diseases (eg, autoimmune diseases as disclosed herein), inflammatory disorders, and / or malignant diseases, and / or their infections, diseases and / or Any disease or disorder or condition associated with malignancy (CVID, other primary immunodeficiencies, HIV disease, CLL, recurrent bronchitis, sinusitis, otitis media, conjunctivitis, pneumonia, hepatitis, meningitis, herpes zoster To treat or prevent (eg, including but not limited to severe herpes zoster) and / or pneumocystis It may be use. Other diseases and injuries that can be prevented, diagnosed, predicted, and / or treated by the fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention include HIV infection, HTLV-BLV infection, lymphopenia Phagocytic bactericidal dysfunction, thrombocytopenia, and hemoglobinuria, including but not limited to.

  In another embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is a non-classifiable immunodeficiency disease (“CVID”; “acquired agamma”). Also known as “globulinemia” and “acquired hypogammaglobulinemia”) or individuals with a subset of this disease are used to treat and / or diagnose.

  In certain embodiments, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is a diagnostic, predictive of cancer or neoplasm, including immune cells or immune tissue-related cancers or neoplasms, Can be used for prevention and / or treatment. Examples of cancers or neoplasms that can be prevented, diagnosed or treated with the albumin fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention include, but are not limited to: Acute myeloid leukemia, chronic myelogenous leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, plasmacytoma, multiple myeloma, Burkitt's lymphoma, EBV-transformed (EBV-transformed) ) Diseases and / or diseases and disorders described in the section entitled “Hyperproliferative disorders” elsewhere herein.

  In another specific embodiment, an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention is used as a therapy to reduce cell proliferation of large B cell lymphoma.

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used as a means to reduce the involvement of B cells and Ig associated with chronic myeloid leukemia. The

  In certain embodiments, the compositions of the invention are used as factors to boost immune responsiveness among B cell immunodeficient individuals (eg, individuals who have undergone partial or complete splenectomy). The

(Blood related disorders)
The albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention can be used to modulate hemostatic activity (stop bleeding) or thrombolytic activity (clot formation). For example, by increasing the hemostatic activity or thrombolytic activity, the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can be used to treat blood coagulation diseases, disorders and / or conditions (eg, Treating or preventing wounds resulting from trauma, surgery or other causes, afibrinogenemia, factor deficiency, hemophilia), blood platelet diseases, disorders and / or conditions (eg, thrombocytopenia) Can do. Alternatively, albumin fusion proteins of the present invention and / or polynucleotides encoding albumin fusion proteins of the present invention that can reduce hemostatic or thrombolytic activity can be used to inhibit or lyse blood clots. These molecules may be important in the treatment or prevention of a heart attack (infarction), stroke or scar.

  In a specific embodiment, the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention is thrombosis, arterial thrombosis, venous thrombosis, thromboembolism, pulmonary embolism, atherosclerosis. , Myocardial infarction, transient ischemic attack, unstable angina, prevention, diagnosis, prediction and / or treatment. In certain embodiments, an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention may be treated with an angioplasty procedure to prevent occlusion of a saphenous vein graft. Prosthetic heart valves and / or caps to reduce the risk of stroke in patients with atrial fibrillation, including non-rheumatic atrial fibrillation, in order to reduce the risk of peripheral thrombosis It can be used to reduce the risk of embolism associated with valve disease. Other uses for albumin fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention include extracorporeal devices (eg, intravascular cannulas, vascular access shunts in hemodialysis patients, hemodialysis Prevention of obstruction in the machine, and cardiopulmonary bypass machine), but is not limited thereto.

  In another embodiment, the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention comprises blood and / or blood-forming organs associated with tissue in which the polypeptide of the present invention is expressed. It can be used for prevention, diagnosis, prediction and / or treatment of diseases and disorders.

  The fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can be used for regulating hematopoietic activity (formation of blood cells). For example, an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention may comprise all or a subset of blood cells (eg, red blood cells, lymphocytes (B cells or T cells), spinal cells (eg, , Basophils, eosinophils, neutrophils, mast cells, macrophages) and platelets). The ability to reduce the amount of blood cells, or a subset of blood cells, can be useful for the prevention, detection, diagnosis and / or treatment of anemia and leukopenia as described below. Alternatively, the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention may comprise all or a subset of blood cells (eg, red blood cells, lymphocytes (B cells or T cells), spinal cells (eg, It can be used to reduce the amount of basophils, eosinophils, neutrophils, mast cells, macrophages) and platelets). The ability to reduce the amount of blood cells, or a subset of blood cells, can be useful in the prevention, detection, diagnosis and / or treatment of leukocytosis (eg, eosinophilia).

  The fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can be used to prevent, treat or diagnose blood cachexia.

  Anemia is a condition in which the number of red blood cells or the amount of hemoglobin (the protein that carries oxygen) is below normal. Anemia can be caused by excessive bleeding, decreased erythropoiesis, or increased red blood cell destruction (hemolysis). The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention may be useful in treating, preventing and / or diagnosing anemia. Examples of anemia that can be treated, prevented or diagnosed by the albumin fusion protein of the present invention and / or a polynucleotide encoding the albumin fusion protein of the present invention include iron deficiency anemia, hypochromia, microcytic anemia, yellowness, genetics Ironblastic anemia, idiopathic acquired ironblastic anemia, erythropoiesis, megaloblastic anemia (eg, pernicious anemia, (vitamin B12 deficiency) and folate deficiency anemia), aplastic anemia Hemolytic anemia (eg, autoimmune hemolytic anemia, microvascular abnormal hemolytic anemia, and paroxysmal nocturnal hemoglobinuria). An albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention may be useful in treating, preventing, and / or diagnosing anemia associated with a disease, and anemia associated with the disease These include, but are not limited to, anemia associated with systemic lupus erythematosus, anemia associated with cancer, anemia associated with lymphoma, anemia associated with chronic kidney disease, and anemia associated with swollen spleen. An albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention can be useful in treating, preventing and / or diagnosing anemia resulting from drug treatment, such as Anemia associated with methyldopa, anemia associated with dapsone, and / or anemia associated with sulfa drugs. Furthermore, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention may be useful in treating, preventing and / or diagnosing anemia associated with abnormal red blood cell structure. Such anemias include, but are not limited to, hereditary spherical erythrocytes, hereditary elliptical erythrocytosis, glucose-6-phosphate dehydrogenase deficiency, and sickle cell anemia.

  The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention is a hemoglobin abnormality (for example, abnormalities associated with sickle cell anemia, hemoglobin C disease, hemoglobin SC disease, and hemoglobin E disease) ) May be useful in treating, preventing, and / or diagnosing. Further, the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention diagnoses thalassemia (including but not limited to major and minor forms of α-thalassemia and β-thalassemia) May be useful in prognosis, prevention and / or treatment.

  In another embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is thrombocytopenic (eg, idiopathic thrombocytopenic purpura and thrombotic thrombocytopenic purpura). ), Von Willebrand disease, hereditary platelet disorders (eg, storage pool disease (eg, Chediak-East and Hermansky-Padlac syndrome), thromboxane A2 dysfunction, thrombophilia, and Bernard-Soulier syndrome), hemolytic uremic syndrome, hemophilia (eg, hemophilia A or factor VII deficiency, and Christmas disease or factor IX deficiency), hereditary hemorrhagic telangiectasia (Randu) -Austler-Weber syndrome and Diagnoses, prognoses, prevents and / or treats bleeding disorders including, but not limited to, allergic purpura (Henoho-Schönlein purpura) and generalized intravascular coagulation syndrome May be useful in some cases.

  The effects of the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention on blood clotting time are as follows: whole blood partial thromboplastin time (PTT), activated partial thromboplastin time (aPTT), active clotting It can be monitored using any coagulation test known in the art, including but not limited to time (ACT), remineralization active clotting time or Lee-White clotting time.

  Some diseases and various drugs can cause platelet dysfunction. Thus, in certain embodiments, an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention is an acquired platelet dysfunction (eg, renal failure, leukemia, multiple myeloma, hepatic Platelet dysfunction associated with cirrhosis and systemic lupus erythematosus, and drug treatment (including treatment with high doses of aspirin, ticlopidine, nonsteroidal anti-inflammatory drugs (used for arthritis, pain, and sprains), and penicillin It may be useful in diagnosing, prognosing, preventing and / or treating platelet dysfunction associated with).

In another embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is characterized by an increase or decrease in white blood cell count or is associated with an increase or decrease in white blood cell count It may be useful in diagnosing, prognosing, preventing and / or treating diseases and disorders. Leukopenia occurs when the white blood cell count decreases below normal. Leukopenia includes, but is not limited to, neutropenia and lymphopenia. An increase in white blood cell count compared to normal is known as leukocytosis. The body produces an increased number of white blood cells during infection. Thus, leukocytosis can simply be a normal physiological parameter that reflects infection. Alternatively, leukocytosis can be an indicator of other diseases such as injury or cancer.
Leukocytoses include, but are not limited to, eosinophilia and macrophage accumulation. In certain embodiments, the albumin fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention may be useful in diagnosing, prognosing, preventing and / or treating leukopenia. In other specific embodiments, albumin fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention are useful in diagnosing, prognosing, preventing and / or treating leukocytosis. obtain.

  Leukopenia can be a state in which all types of leukocytes are generally depleted, or can be a specific depletion of a particular type of leukocytes. Accordingly, in certain embodiments, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention diagnoses and prognoses a decrease in neutrophil count (known as neutropenia). May be useful in preventing and / or treating. Neutropenia that can be diagnosed, prognosticated, prevented and / or treated with an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention includes, but is not limited to: Not: resulting from or infant infant genetic agranulocytosis, familial neutropenia, periodic neutropenia, dietary deficiencies (eg vitamin B12 deficiency or folate deficiency) Or results from related neutropenia, drug treatment (eg, antibiotic regimens (eg, penicillin treatment), sulfonamide treatment, anticoagulant treatment, antispasmodic drugs, antithyroid drugs, and cancer chemotherapy) Treatment-related neutropenia and some bacteria Favorable conditions that may arise in connection with infection or viral infections, allergic disorders, autoimmune diseases, individuals with swollen spleens (eg, Ferti syndrome, malaria, and sarcoidosis) and with several drug treatment regimens Neutropenia resulting from increased neutrophil destruction.

  The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention is stress, drug treatment (eg, drug treatment with corticosteroids, cancer chemotherapy, and / or radiation therapy), AID infection , And / or other diseases (eg, cancer, rheumatoid arthritis, systemic lupus erythematosus, chronic infections, some viral infections, and / or genetic disorders (eg, Di George syndrome, Viscott-Oldrich syndrome, (Such as severe combined immunodeficiency, telangiectasia ataxia)) or related lymphopenia, including but not limited to lymphocytopenia (B and / or T lymphocytes) When diagnosing, prognosing, preventing and / or treating It may be in use.

  The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention includes, but is not limited to, Goshe disease, Niemann-Pick disease, Letterer-Give disease, and Hand-Schuller-Christian disease. It may be useful in diagnosing, prognosing, preventing and / or treating diseases and disorders associated with macrophage count and / or macrophage function.

  In another embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention comprises idiopathic hypereosinophilic syndrome, eosinophilia-myalgia syndrome, and hand- It may be useful in diagnosing, prognosing, preventing and / or treating diseases and disorders related to eosinophil count and / or eosinophil function, including but not limited to Schuler-Christian disease.

  In yet another embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is acute lymphocytic (lymphoblastic) leukemia (ALL), acute myeloid (myeloid ( myelocytic, myelogenous, myeloblastic or myelomonocytic leukemia, chronic lymphocytic leukemia (eg, B cell leukemia, T cell leukemia, Sezary syndrome, and hairy cell leukemia), chronic myeloid (myeloid) Diagnose and prognose leukemia and lymphoma including but not limited to myeloid, myelogenous or granulocytic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, and mycosis fungoides Yes in prevention and / or treatment It can be in.

  In other embodiments, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is a plasma cell dysplasia, monoclonal hypergammaglobulinemia, monoclonality of unknown significance. Plasma cells including, but not limited to, monoclonal gammopathies of undetermined signature, multiple myeloma, macroglobulinemia, Waldenstrom macroglobulinemia, cryoglobulinemia, and Lehno phenomenon May be useful in diagnosing, prognosing, preventing and / or treating other diseases and disorders.

  In another embodiment, the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention is polycythemia vera, relative erythrocytosis, secondary polycytheemia, Bone marrow, including but not limited to myelofibrosis, acute myelofibrosis, myeloid metaplasia of unknown cause, thrombocythemia (including both primary and secondary thrombocythemia), and chronic myelogenous leukemia It may be useful in treating, preventing and / or diagnosing a proliferative disorder.

  In other embodiments, albumin fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention may be useful as a treatment to increase blood cell production prior to surgery.

  In other embodiments, an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention comprises neutrophils, phagocytosis, superoxide production, antibody-dependent cytotoxicity, eosinophils and It may be useful as an agent for enhancing macrophage migration.

  In other embodiments, an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention may be useful as an agent to increase the number of stem cells in the circulatory system prior to stem cell pheresis. . In another specific embodiment, an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention is useful as an agent to increase the number of stem cells in the circulatory system prior to plateletpheresis. possible.

  In other embodiments, an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention may be useful as an agent to increase cytokine production.

  In other embodiments, albumin fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention may be useful in preventing, diagnosing and / or treating primary hematopoietic disorders.

(Hyperproliferative disorder)
In certain embodiments, the fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention can be used to treat or detect hyperproliferative disorders, including neoplasias. An albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention may inhibit the growth of a disorder through direct or indirect interactions. Alternatively, an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention can proliferate other cells that can inhibit hyperproliferative disorders.

  For example, hyperproliferative disorders can be treated by increasing the immune response, in particular by increasing the antigenic quality of the hyperproliferative disorder or by proliferating, differentiating or mobilizing T cells. This immune response can be increased by either enhancing an existing immune response or initiating a new immune response. Alternatively, reducing the immune response can also be a method of treating a hyperproliferative disorder (eg, a chemotherapeutic agent).

  Examples of hyperproliferative disorders that can be treated or detected by an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention include, but are not limited to: colon, abdomen, Bone, breast, digestive system, liver, pancreas, peritoneum, endocrine gland (upper renal, parathyroid, pituitary, testis, ovary, thymus, thyroid), eye, head and neck, nerves (central and peripheral), lymph Neoplasm located in the system, pelvis, skin, soft tissue, spleen, rib cage, and urinary tract.

  Similarly, other hyperproliferative disorders can also be treated or detected by albumin fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention. Examples of such hyperproliferative disorders include but are not limited to: acute childhood lymphoblastic leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, adrenal gland Cortical carcinoma, adult (primary) hepatocellular carcinoma, adult (primary) liver cancer, adult acute lymphocytic leukemia, adult acute myeloid leukemia, adult Hodgkin's disease, adult Hodgkin lymphoma, adult lymphocytic leukemia Adult non-Hodgkin lymphoma, adult primary liver cancer, adult soft tissue sarcoma, AIDS-related lymphoma, AIDS-related malignant disease, anal cancer, astrocytoma, bile duct cancer, bladder cancer, bone cancer, brain stem glioma, brain tumor, breast cancer, Cancer of the renal pelvis and ureter, central nervous system (primary) lymphoma, central nervous system lymphoma, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, pediatric (Primary) hepatocellular carcinoma, pediatric (primary) liver cancer, pediatric acute lymphoblastic leukemia, pediatric acute myeloid leukemia, pediatric brain stem glioma, pediatric cerebellar astrocytoma, pediatric cerebral astrocytoma Cytomas, Childhood Extracranial Germ Cell Tumor, Pediatric Hodgkin's Disease, Pediatric Hodgkin Lymphoma, Pediatric Hypothalamic and Visceral Glioma Leukemia, pediatric medulloblastoma, pediatric non-Hodgkin lymphoma, pediatric pineal gland and tent undifferentiated neuroectodermal tumor, pediatric primary liver cancer, pediatric rhabdomyosarcoma, pediatric soft tissue sarcoma, children Optic tract and hypothalamic glioma, chronic lymphocytic white blood Chronic myelogenous leukemia, colon cancer, cutaneous T-cell lymphoma, endocrine pancreatic islet cell carcinoma, endometrial cancer, ependymoma, epithelial cancer, esophageal cancer, Ewing sarcoma and related tumors, Exocrine Pancreatic Cancer, skull External germ cell tumor (Extracranial Germ Cell Tumor), extragonadal germ cell tumor (Extragonadal Ger Cell Tumor), extrahepatic cholangiocarcinoma, ocular cancer, female breast cancer, goche disease, gallbladder cancer, gastric cancer, gastrointestinal carcinoma, gastrointestinal tumor, Germ cell tumor, gestational trophoblastic tumor, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, Hodgkin disease, Hodgkin lymphoma, hypergammaglobulinemia, hypopharyngeal cancer, intestine Cancer, intraocular melanoma, islet cell carcinoma, islet cell pancreatic cancer, capage sarcoma, kidney cancer, laryngeal cancer, lip and oral cancer, liver cancer, lung cancer, lymphoproliferative disorder, macroglobulinemia, Male breast cancer, malignant mesothelioma, malignant thymoma, medulloblastoma, melanoma, mesothelioma, metastatic occult primary squamous neck cancer, metastatic primary squamous cervical cancer (Metastatic Primary Squamous Neck Cancer), Metastatic Squamous Neck Cancer, Multiple Myeloma, Multiple Myeloma / Plasma Cell Neoplasia, Myelodysplastic Syndrome, Myelogenous Leukemia (Myelogenus) Leukemia), myeloid leukemia, myeloproliferative disorders, nasal and sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma in pregnancy, non-melanoma skin cancer, non-melanoma skin cancer Small cell lung cancer, occult primary metastatic squamous neck cancer, oropharyngeal cancer, bone / malignant fibrosarcoma, osteosarcoma / malignant fibrous histiocytoma, bone osteosarcoma / malignant Fibrous histiocytoma, ovarian epithelial cancer, ovarian germ cell tumor, ovarian borderline tumor, pancreatic cancer, paraproteinemia, purpura, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumor, plasma cell neoplasm / Multiple myeloma, primary central nervous system lymphoma, primary liver cancer, prostate cancer, rectal cancer, Cell carcinoma, renal pelvic and ureteral cancer, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, sarcoidosis sarcoma, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cervical cancer, stomach cancer, tent Upper undifferentiated neuroectodermal and pineal tumors, T cell lymphoma, testicular cancer, thymoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, transitional renal pelvic and ureteral cancer, trophoblastic tumor , Ureteral and renal pelvic cell cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, optic duct and hypothalamic glioma, vulvar cancer, Waldenstrom macroglobulinemia, Wilms tumor, and any Other hyperproliferative diseases and neoplasias that are positioned in the organ systems listed above.

  In another preferred embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used to diagnose, prognose, prevent and / or treat premalignant conditions, and as described above. Prevention of progression to a neoplastic or malignant condition, including but not limited to such disorders. Such use is indicated for neoplastic or cancerous (especially where hyperplasia, metaplasia, or most particularly dysplastic non-neoplastic cell proliferation occurs (such abnormal For a review of proliferative states, see Robbins and Angell, 1976, Basic Pathology, 2nd edition, WB Saunders Co., Philadelphia, pp. 68-79)), known or suspected In the situation.

  Hyperplasia is a controlled form of cell proliferation that involves an increase in the number of cells in a tissue or organ without significant changes in structure or function. Examples of the hyperplastic disorder that can be diagnosed, prognosticated, prevented and / or treated using the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention include the following. Without limitation: Vascular follicular mediastinal lymph node proliferation, eosinophilia associated vascular lymphoid hyperplasia, atypical melanocyte hyperplasia, basal cell hyperplasia, benign giant lymph node proliferation, cementum hyperplasia, congenital Adrenal hyperplasia, congenital hyperplasia, cystic proliferation, breast cystic hyperplasia, denture fibrosis, ductal hyperplasia, endometrial hyperplasia, fibromuscular hyperplasia, local epithelial thickening, gingival proliferation, Inflammatory fibrous hyperplasia, inflammatory papillary hyperplasia, intravascular papillary endothelial hyperplasia, nodular prostate hyperplasia, nodular regenerative hyperplasia, pseudoepithelioma growth, senile sebaceous hyperplasia, and epilepsy Sex thickening.

  Metaplasia is a controlled form of cell proliferation in which one type of adult cell or fully differentiated cell replaces another type of adult cell. Examples of metaplasia disorders that can be diagnosed, prognosticated, prevented and / or treated using the albumin fusion protein of the present invention and / or a polynucleotide encoding the albumin fusion protein of the present invention include the following. Without limitation: Unexplained myeloid metaplasia, apocrine metaplasia, atypical metaplasia, autogenous metaplasia, connective tissue metaplasia, epithelial metaplasia, intestinal metaplasia, metaplastic anemia, deformed ossification, Dysplastic polyps, myeloplasia, primary myeloid metaplasia, secondary myelogenic metaplasia, flattening, amniotic flattening, and symptomatic myelogenous.

  Dysplasia is frequently a precursor to cancer and is mainly found in the epithelium; dysplasia is the most disturbed form of non-neoplastic cell proliferation, which is a loss of individual cell uniformity and Including loss of cellular architectural orientation. Dysplastic cells often have abnormally large, deeply stained nuclei and are pleomorphic. Dysplasia occurs characteristically in the presence of chronic hypersensitivity or inflammation. Examples of dysplasia disorders that can be diagnosed, prognosticated, prevented and / or treated using the fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention include, but are not limited to: Not: sweatless ectodermal dysplasia, anteroposterior dysplasia, choking thoracic dysplasia, atrial digital dysplasia, bronchopulmonary dysplasia, telencephalon dysplasia, cervical dysplasia, cartilage ectodermal Dysplasia, clavicle skull dysplasia, congenital ectodermal dysplasia, skull stem dysplasia, skull carpal and tarsal bone dysplasia, skull metaphyseal dysplasia, dentine dysplasia, diaphyseal dysplasia, ectoderm Sexual dysplasia, enamel dysplasia, brain-ophthalmic dysplasia, tarsal bone hypertrophy, multiple Abnormal dysplasia, punctate apical dysplasia, epithelial dysplasia, facial genital genital dysplasia, familial jaw bone fibrosis, familial white wing dysplasia, fibromuscular dysplasia, fibrous bone dysplasia Flowering osteodysplasia, hereditary renal-retinal dysplasia, sweating ectodermal dysplasia, anhidrotic ectodermal dysplasia, lymphocytopenic thymic dysplasia, Breast dysplasia, maxillofacial dysplasia, metaphyseal dysplasia, Mondini inner ear dysplasia, single fibrosis dysplasia, mucosal epithelial dysplasia, multiple epiphyseal dysplasia, ocular ear spinal dysplasia, ocular finger formation Abnormal, Ophthalmic spine dysplasia, Tooth dysplasia, Ocular mandibular limb dysplasia, Apical cementum dysplasia, Multiple fibrous bone dysplasia, Pseudochondral dysplasia vertebral tip dysplasia, Retinal dysplasia, Medium - Visual dysplasia, vertebral end dysplasia, and ventricular radial dysplasia.

  Further, examples of the preneoplastic disorder that can be diagnosed, prognosticated, prevented and / or treated with the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention include the following: Not limited to: benign abnormal proliferative disorders (eg benign tumor, fibrocystic condition, tissue hypertrophy, intestinal polyp, colon polyp, and esophageal dysplasia), vitiligo, keratosis, Bowen's disease, farmer's skin, sunlight lips Flame, and actinic keratosis.

  In another embodiment, albumin fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention can be used to diagnose and / or prognose disorders related to tissues in which the polypeptide is expressed.

  In another embodiment, the present specification uses an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention conjugated to a toxin or radioisotope as described herein. Cancers and neoplasms can be treated, including but not limited to those described in the document. In a further preferred embodiment, acute bone marrow using an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention conjugated to a toxin or radioisotope as described herein. Sexual leukemia can be treated.

  Furthermore, the albumin fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention can affect apoptosis and are therefore useful in treating many diseases related to increased cell survival or inhibition of apoptosis. . For example, the diseases relating to the increase in cell survival or inhibition of apoptosis that can be diagnosed, prognosticated, prevented and / or treated using the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention , Including: cancer (eg, follicular lymphoma, cancer with p53 mutation, and hormone-dependent tumor (including but not limited to: colon cancer, cardiac tumors, pancreatic cancer, Melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, gastric cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelial tumor, osteoblastoma, giant cell tumor, osteosarcoma, cartilage Sarcoma, adenoma, breast cancer, prostate cancer, capage sarcoma, and ovarian cancer)); autoimmune disorders (eg, multiple sclerosis, Sjogren's syndrome, Thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus, and immune-related glomerulonephritis and rheumatoid arthritis and viral infections (eg , Herpesviruses, poxviruses and adenoviruses) inflammation, graft versus host disease, acute graft rejection, and chronic graft rejection.

  In preferred embodiments, the fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention are used to inhibit the growth, progression, and / or metastasis of cancer (especially those listed above).

  Further diseases or conditions relating to increasing cell survival that can be diagnosed, prognosticated, prevented and / or treated with an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention include: The following progression and / or metastasis may include, but are not limited to: malignant tumors and related disorders (eg, leukemia (acute leukemia (eg, acute lymphocytic leukemia, acute myeloid leukemia (myeloblastic leukemia, pre- Including myelocytic leukemia, myelomonocytic leukemia, monocytic leukemia and erythroleukemia), and chronic leukemia (eg, chronic myelogenous leukemia (granulocytic leukemia) and chronic lymphocytic leukemia)) Erythrocytic hyperplasia, lymphoma (eg, Hodgkin's disease and Hodgkin's disease), multiple myeloma, Waldenstrom macroglobulinemia, heavy chain disease, and solid tumors, including but not limited to: Sarcomas and cancers (eg, fibrosarcoma, mucus Sarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, hemangiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphatic endothelial, periosteum, mesothelioma, Ewing's tumor, leiomyosarcoma, lateral Rhabdomyosarcoma, colon cancer, pancreas, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary gland carcinoma, cystadenocarcinoma, medullary carcinoma , Bronchial primordial cancer, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, fetal cancer, Wilmsoma, cervical cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, stellate Cell tumor, medulloblastoma Craniopharyngioma, ventricular ependymoma, pineal gland, hemangioblastoma, acoustic neuroma, oligodendroma, meningioma, melanoma, neuroblastoma As well as retinoblastoma)).

  Diseases associated with increasing apoptosis that can be diagnosed, prognosticated, prevented and / or treated with an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention include: AIDS; Neurodegenerative disorders (eg, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration, and brain tumors or primary associated diseases); autoimmune disorders (eg, multiple diseases) Sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus, and immune-related glomerulonephritis ulonephritis) and rheumatoid arthritis), myelodysplastic syndromes (eg, aplastic anemia), graft versus host disease, ischemic disorders (eg, due to myocardial infarction, stroke, and reperfusion injury), liver Disorders (eg, hepatic disorders associated with hepatitis, ischemia / reperfusion disorders, cholestasis (bile duct disorders), and liver cancer); toxin-induced liver diseases (eg, due to alcohol), septic Shock, cachexia and loss of appetite.

  Hyperproliferative diseases and / or disorders that can be diagnosed, prognosticated, prevented and / or treated with an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention include: But not limited to: liver, abdomen, bone, breast, digestive system, pancreas, peritoneum, endocrine gland (adrenal gland, parathyroid, pituitary, testis, ovary, thymus, thyroid), eye, head, and Neoplasm located in the neck, nervous system (central and peripheral nerves), lymphatic system, pelvis, skin, parenchyma, spleen, thorax and urinary tract.

  Similarly, other hyperproliferative disorders can also be diagnosed, prognosticated, prevented and / or treated with the albumin fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention. Examples of such hyperproliferative disorders include, but are not limited to: hypergammaglobulinemia, lymphoproliferative disorder, paraproteinemia, purpura, sarcoidosis, Sezary syndrome, Waldenstrom In addition to macroglobulinemia, Gossy disease, histiocytosis, and neoplasia, any other hyperproliferative disease located in the organ system listed above.

  Another preferred embodiment utilizes the polynucleotides of the invention to inhibit abnormal cell division by gene therapy using the invention and / or protein fusions or fragments thereof.

  Accordingly, the present invention provides a method for treating a cell proliferative disorder by inserting a polynucleotide of the present invention into an abnormally proliferating cell, wherein the polynucleotide suppresses its expression. .

Another embodiment of the invention provides a method of treating a cell proliferative disorder in an individual, wherein the method administers one or more active gene copies of the invention to abnormally proliferating cell (s). The process of carrying out is included. In a preferred embodiment, the polynucleotide of the invention is a DNA construct comprising a recombinant expression vector that is effective in expressing a DNA sequence encoding the polynucleotide. In another preferred embodiment of the invention, the DNA construct encoding the fusion protein of the invention is inserted into a cell to be treated using a retrovirus (or more preferably an adenoviral vector) ( See GJ Nabel et al., PNAS 1999 96: 324-326, incorporated herein by reference). In the most preferred embodiment, the viral vector is defective and does not transform non-proliferating cells, only transforming cells. Furthermore, in a preferred embodiment, the polynucleotide of the invention inserted into a proliferating cell, either alone, in combination with other polynucleotides, or fused to other polynucleotides is then Can be regulated via external stimuli (ie, administration of magnetism, specific small molecules, chemicals or drugs, etc.), which external stimuli can induce the expression of the encoded protein product Acts on upstream promoters.
Thus, the beneficial therapeutic effects of the present invention can be clearly modulated (ie, increase, decrease or inhibit the expression of the present invention) based on this external stimulus.

  The polynucleotides of the present invention may be useful in suppressing the expression of neoplastic genes or antigens. “Suppressing the expression of a neoplastic gene” means suppression of transcription of this gene, destruction of this gene transcript (pre-message RNA), inhibition of splicing, destruction of messenger RNA, interference with post-translational modification of protein, protein Intended to disrupt the normal function of the protein.

  For topical administration to abnormally proliferating cells, the polynucleotides of the invention can be administered by any method known to those of skill in the art. This method includes transfection, electroporation, cell microinjection, or a vehicle such as a liposome, lipofection, or naked polynucleotide, or any other method described throughout this specification. However, it is not limited to these. The polynucleotides of the present invention may be obtained from retroviral vectors known to those of skill in the art (Gilboa, J. Virology 44: 845 (1982); Hocke, Nature 320: 275 (1986); Wilson et al., Proc. Natl. Acad. Sci. U. S.A. 85: 3014), vaccinia virus system (Chakrabarty et al., Mol. Cell Biol. 5: 3403 (1985), or other efficient DNA delivery systems (Yates et al., Nature 313: 812 (1985)). Which can be delivered by known gene delivery systems such as, but not limited to, these references are exemplary only and are incorporated herein by reference. Specific delivery or transfection , And utilize retrovirus or adenovirus (as described in the art and described elsewhere herein) delivery systems known to those skilled in the art to leave non-dividing cells Preferably, host DNA replication is required to incorporate retroviral DNA, and retroviruses cannot replicate autonomously because they lack the retroviral genes required for their life cycle. Utilizing such a retroviral delivery system for polynucleotides targets this gene and construct to abnormally proliferating cells and leaves normal cells that are not dividing.

  The polynucleotides of the invention can be directly applied to the site of cell proliferative disorder / disease in internal organs, body cavities, etc. by using an imaging device that is used to guide the needle directly to the diseased site. Can be delivered to. The polynucleotides of the invention can also be administered to the disease site at the time of surgical intervention.

  A “cell proliferative disorder”, whether benign or malignant, is any organ, cavity, or body part characterized by a single or multiple local abnormal growth of cells, groups of cells, or tissues By any human or animal disease or disorder affecting one or any combination.

  Any amount of the polynucleotides of the invention can be administered as long as they have a biologically inhibiting effect on the growth of the treated cells. Furthermore, it is possible to administer more than one polynucleotide of the invention simultaneously to the same site. “Biologically inhibit” means partial or total growth inhibition, as well as a decrease in the rate of cell proliferation or growth. Biologically inhibitory doses are determined by assessing the effect of a polynucleotide of the invention on target malignant or abnormal growth cell growth in tissue culture, tumor growth in animals and cell cultures, Or it can be determined by any other method known to those skilled in the art.

  Furthermore, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention can be used alone, as a protein fusion or directly as described elsewhere herein. Useful in inhibiting angiogenesis of proliferating cells or tissues, either in combination with other polypeptides, either intentionally or indirectly. In the most preferred embodiment, this anti-angiogenic effect can be achieved indirectly, eg, through inhibition of hematopoietic tumor-specific cells (eg, tumor-associated macrophages) (Joseph IB et al., J Natl Cancer Inst, 90 (21): 1648-53 (1998), which is incorporated herein by reference).

  An albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention may be useful in inhibiting proliferating cells or tissues through induction of apoptosis. These fusion proteins and / or polynucleotides include, for example, death-domain receptors (eg, tumor necrosis factor (TNF) receptor-1, CD95 (Fas / APO-1), TNF receptor related apoptosis mediating proteins (TRAMP) and TNF-related apoptosis-inducing ligand (TRAIL) receptor-1 and -2 (Schulze-Osthoff K et al., Eur J Biochem 254 (3): 439-59 (1998), incorporated herein by reference. ))))) Can act either directly or indirectly to induce proliferative cell and tissue apoptosis. Furthermore, in another preferred embodiment of the invention, these fusion proteins and / or polynucleotides may be through other mechanisms, such as in the activation of other proteins that activate apoptosis, alone or as small molecule drugs or Adjuvants (eg, apoptonin, galectins, thioredoxins, anti-inflammatory proteins (eg, Mutat Res 400 (1-2): 447-55 (1998), Med Hypotheses. 50 (5): 423-33) (1998), Chem Biol Interact. Apr 24; 111-112: 23-34 (1998), J Mol Med. 76 (6): 402-12 (1998), Int J Tissue React; 1): 3-15 (1998) (all of which are incorporated herein by reference))) in combination with any of the above, through stimulating the expression of this protein, It can induce apoptosis.

  The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention is useful in inhibiting metastasis of proliferating cells or tissues. Inhibition may be as a direct result of administering these albumin fusion proteins and / or polynucleotides, or indirectly (eg, inhibiting metastasis, as described elsewhere herein. Can be activated (see, eg, Curr Top Microbiol Immunol 1998; 231: 125-41, which is incorporated herein by reference). Such therapeutic effects of the present invention can be achieved either alone or in combination with small molecule drugs or adjuvants.

  In another embodiment, the present invention provides a composition comprising an albumin fusion protein of the present invention and / or a polynucleotide encoding an albumin fusion protein of the present invention to this protein bound by the albumin fusion protein of the present invention. Methods are provided for delivery to targeted cells that bind or express a polypeptide associated with the protein. The albumin fusion proteins of the present invention can be linked to heterologous polypeptides, nucleic acids, toxins, or prodrugs through hydrophobic, hydrophilic, ionic and / or covalent interactions.

  The albumin fusion proteins of the present invention are directed against proliferative antigens and immunogens directly (eg, when the albumin fusion proteins of the present invention are “vaccinated”) to respond to the antigens and immunogens described above. Immunity of proliferating cells or tissues, either in response to an immune response) or indirectly (eg, in activating expression of a protein (eg, chemokine) known to enhance the immune response) Useful in enhancing protogenicity and / or antigenicity.

(Kidney disorder)
The albumin fusion protein of the invention and / or polynucleotide encoding the albumin fusion protein of the invention may be used to treat, prevent, diagnose and / or prognose a disorder of the renal system. Kidney disorders that can be diagnosed, prognosticated, prevented, and / or treated using the compositions of the present invention include, but are not limited to: renal failure, nephritis, renal vascular disorders, metabolic kidneys Disorders and congenital kidney disorders, kidney urine disorders, autoimmune disorders, sclerosis and necrosis, electrolyte inequalities, and kidney cancer.

  Renal diseases that can be diagnosed, prognosticated, prevented, and / or treated using the compositions of the present invention include, but are not limited to: acute renal failure, chronic renal failure, atheroembolism, end stage Kidney disease, renal inflammatory disease (eg, acute glomerulonephritis, post-infection glomerulonephritis, rapidly progressive glomerulonephritis, nephrotic syndrome, membranous glomerulonephritis, familial nephrotic syndrome, membranoproliferative glomerulonephritis I and II, mesangial proliferative glomerulonephritis, chronic glomerulonephritis, acute tubulointerstitial nephritis, chronic tubulointerstitial nephritis, acute streptococcal glomerulonephritis (PSGN), pyelonephritis, lupus nephritis, Chronic nephritis, interstitial nephritis, and post-streptococcal glomerulonephritis), renal vascular disorders (eg, renal infection, atherothrombotic kidney disease, cortical necrosis, malignant nephrosclerosis, renal vein thrombosis Renal damage resulting from renal underperfusion, renal retinopathy, renal ischemia-reperfusion, renal artery embolism, and renal artery stenosis, such as renal artery (eg, renal artery stenosis) Pyelonephritis, hydronephrosis, urolithiasis (nephrolithiasis, nephrolithiasis), reflux nephropathy, ureteral infection, urinary retention, and acute or chronic unilateral obstructive urinary tract disease.

  Further, using the composition of the present invention, renal metabolic disorders and congenital disorders (eg, uremia, renal amyloidosis, renal dysplasia, tubular acidosis, renal diabetes, nephrogenic diabetes insipidus) Cystinuria, Fanconi syndrome, renal rickets (renal rickets), Hartnup disease, Barter syndrome, Riddle syndrome, polycystic kidney disease, medullary cyst disease, medullary sponge kidney, Alport syndrome, Nail-patella syndrome, congenital nephrotic syndrome, crush syndrome, horseshoe kidney, diabetic neuropathy, nephrogenic diabetes insipidus, analgesic nephropathy, kidney stones and membranous nephropathy), and renal autoimmune disorders ( For example, systemic lupus erythematosus (SLE), Goodpasture syndrome, IgA nephropathy, and IgM Ngiumu proliferative glomerulonephritis)) diagnosing, prognosis, prevention, and / or may be treated.

  The compositions of the present invention may also be used to diagnose, prognose, prevent, and / or treat the following: sclerosing or necrotic disorders of the kidney (eg, glomerulosclerosis, diabetic nephropathy, focal Segmental glomerulosclerosis (FSGS), necrotizing glomerulonephritis, and renal papillary necrosis), renal cancer (eg, nephroma, adrenal gland, nephroblastoma, renal cell carcinoma, transitional cell carcinoma, renal adenocarcinoma) , Squamous cell carcinoma, and Wilmsoma), and electrolyte inequalities (eg, renal calcification, abscess, edema, hydronephrosis, proteinuria, hyponatremia, hypernatremia, hypokalemia, hyperkalemia, Hypocalcemia, hypercalcemia, hypophosphatemia, and hyperphosphatemia).

  The compositions of the invention can be administered using any method known in the art, including direct needle injection, intravenous injection, topical administration, catheter injection, particle gun at the delivery site. (Biolistic) injections, particle accelerators, gel foam sponge depots, other commercially available depot materials, osmotic pumps, oral or suppository solid pharmaceutical formulations, decanting or topical application during surgery, aerosol delivery, It is not limited to these. Such methods are known in the art. The compositions of the present invention may be administered as part of a therapeutic agent, described in more detail below. Methods for delivering polynucleotides are described in more detail herein.

(Cardiovascular disorder)
Treating and preventing cardiovascular disorders including, but not limited to, peripheral arterial disease such as limb ischemia using the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention, Diagnosis and / or prognosis can be made.

  Cardiovascular disorders include cardiovascular abnormalities such as arterio-arterial fistula, arteriovenous fistula, cerebral arteriovenous congenital anomalies, congenital heart defects, pulmonary valve atresia, and scimitar syndrome However, it is not limited to these. Congenital heart defects include aortic constriction, triaxial heart, coronary vascular anomalies, crossed heart, right thoracic heart, patent ductus arteriosus, Ebstein malformation, Eisenmenger complex, left ventricular dysgenesis syndrome, left thoracic heart, tetralogy of Fallot, transposition of large blood vessels, biventricular right ventricular origin, tricuspid atresia, arterial vessel remnant, and septal defect (Heart septal defects) (e.g., aortopulmonary septal defect), endocardial floor deficiency, lytanbache syndrome, trilogy of Fallot, ventricular heart septal defects They include, but are not limited to.

  Cardiovascular disorders also include arrhythmia, carcinoid heart disease, high cardiac output, low cardiac output, cardiac tamponade, endocarditis (including bacterial), heart Aneurysm, cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, cardiac hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, post-infarction heart rupture , Ventricular septal rupture, heart valve disease, myocardial disease, myocardial ischemia, endocardial effusion, epicarditis (including infarction and tuberculosis), pneumoperiosis, postpericardiotomy syndrome, right heart disease , Rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy complication s), heart disease such as, but not limited to, scimitar syndrome, cardiovascular syphilis, and cardiovascular tuberculosis.

  Arrhythmias include sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia, extrasystole, Adams-Stokes syndrome, leg block, sinoatrial block, long QT syndrome, minor contraction, Lone-Gangong -Include but are not limited to Levine syndrome, Mahaim-type pre-exclusion syndrome, Wolf-Parkinson-White syndrome, sinus failure syndrome, tachycardia, and ventricular fibrillation. Tachycardia includes paroxysmal tachycardia, supraventricular tachycardia, ventricular proper rhythm promotion, atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia, ectopic junction tachycardia, Examples include sinoatrial nodal reentry tachycardia, sinus tachycardia, torsade de pointe, and ventricular tachycardia.

  Heart valve diseases include aortic dysfunction, aortic stenosis, heart murmurs, aortic valve prolapse, mitral valve prolapse, tricuspid prolapse, mitral valve dysfunction, mitral valve Examples include stenosis, pulmonary valve atresia, pulmonary valve dysfunction, pulmonary valve stenosis, tricuspid atresia, tricuspid valve dysfunction, and tricuspid stenosis.

  Myocardial diseases include alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, subvalvular aortic stenosis, subvalvular pulmonary stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial fiber elasticity Disease, endocardial myocardial fibrosis, Keynes syndrome, myocardial reperfusion injury, and myocarditis.

  Myocardial ischemia includes, but is not limited to, coronary artery disease such as angina pectoris, coronary aneurysm, coronary atherosclerosis, coronary artery thrombosis, coronary vasospasm, myocardial infarction, and myocardial stunning.

  Cardiovascular diseases also include aneurysms, angiogenesis abnormalities, hemangiomatosis, bacterial hemangioma symptoms, Hippel-Lindau disease, Krippel-Tornonnay-Weber syndrome, Sturge-Weber syndrome, vasomotor nerve Edema, aortic disease, Takayasu arteritis, aortitis, Lleish syndrome, arterial occlusion disease, arteritis, arteritis, nodular polyarteritis, cerebrovascular disorder, diabetic vascular disorder, diabetic retina , Embolism, thrombosis, acromegaly, sputum, hepatic vein occlusion disorder, hypertension, hypotension, ischemia, peripheral vascular disease, phlebitis, pulmonary vein occlusion disease, Lehno's disease, CREST syndrome, retinal vein occlusion, Scimitar syndrome, superior vena cava syndrome, telangiectasia, telangiectasia ataxia tasia), hereditary hemorrhagic telangiectasia, varicocele, dilated serpentine vein, varicose ulcer, vasculitis, and venous dysfunction.

  Aneurysms include dissecting aneurysms, pseudoaneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, cardiac aneurysms, and iliac aneurysms However, it is not limited to these.

  Arterial occlusive diseases include arteriosclerosis, intermittent claudication, carotid stenosis, fibromuscular dysplasia, mesenteric vascular occlusion, moyamoya disease, renal artery occlusion, retinal arterial occlusion, and obstructive thrombotic vasculitis However, it is not limited to these.

  Cerebrovascular disorders include carotid artery disease, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous congenital abnormality, cerebral artery disease, cerebral embolism and thrombosis, carotid artery thrombus Disease, sinus thrombosis, Wallenberg syndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma, subarachnoid hemorrhage, cerebral infarction, cerebral ischemia (including transient), subclavian artery steal Examples include, but are not limited to, blood syndrome, pericellular leukomalacia, vascular headache, cluster headache, migraine, and vertebra basal dysfunction.

  Embolisms include, but are not limited to, air embolism, amniotic fluid embolism, cholesterol embolism, toe cyanosis syndrome, fat embolism, pulmonary embolism, and thromboembolism. Thrombosis includes, but is not limited to, coronary artery thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid artery thrombosis, sinus thrombosis, Wallenberg syndrome, and thrombophlebitis.

  Ischemic disorders include cerebral ischemia, ischemic colitis, compartment syndrome, anterior compartment syndrome, myocardial ischemia, reperfusion injury, and peripheral limb ischemia. It is not limited to. Examples of vasculitis include aortitis, arteritis, Behcet syndrome, Churg-Strauss syndrome, mucocutaneous lymph node syndrome, obstructive thrombovascular vasculitis, hypersensitivity vasculitis, Schönlein-Henoch purpura (Schoonlein- Henoch purpura), allergic dermatovasculitis and Wegener's granulomatosis include, but are not limited to.

  The albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention can be administered using any method known in the art, including directly at the delivery site. Needle injection, intravenous injection, topical administration, catheter injection, biolistic injection, particle accelerator, gelfoam sponge depot, other commercial depot materials, osmotic pump, oral or suppository solid pharmaceutical formulation, surgery Including, but not limited to, decanting or topical application, aerosol delivery. Such methods are known in the art. Methods for delivering polynucleotides are described in more detail herein.

(Respiratory disorder)
The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can be used to treat, prevent, diagnose and / or prognose respiratory diseases and / or disorders.

  Respiratory diseases and disorders include, but are not limited to: nasal vestitis, non-allergic rhinitis (eg, acute rhinitis, chronic rhinitis, atropine-like rhinitis, vasomotor rhinitis), nasal polyps Sinusitis, juvenile hemangiofibroma, nasal cancer and juvenile papilloma, vocal cord polyp, vocal cord nodules (singer nodules), contact ulcer, vocal cord paralysis, laryngeal emphysema, pharyngitis (eg, viral and bacterial) ), Tonsillitis, tonsillitis, parapharyngeal gap abscess, laryngitis, laryngeal emphysema, and pharyngeal cancer (eg, nasopharyngeal cancer, tonsillar cancer, pharyngeal cancer), lung cancer (eg, squamous cell carcinoma, small Cytomas (oat cell tumors), large cell tumors, and adenocarcinoma), allergic disorders (eosinophilic pneumonia, hypersensitivity pneumonia (eg, exogenous allergic alveolitis, allergic interstitial pneumonia, organic dust lung) Disease, allergic Pulmonary aspergillosis, asthma, Wegener's granulomatosis (granulomatous vasculitis), Goodpascher syndrome), pneumonia (eg, Streptococcus pneumoniae, Staphylococcus aureus), Staphylococcus aureus, Gram-negative bacterial pneumonia (eg, caused by Klebsiell and Pseudomas spp.), Mycoplasma pneumoniae pneumonia, Hemophilus influenzae pneumonia, Legionella pneumophila (legionella disease), and Chlamy virus Chickenpox (varicelle la)).

  Additional diseases and disorders of the respiratory system include but are not limited to: bronchiolitis, polio, croup, RS virus infection, mumps, infectious erythema (fifth disease), childhood rash, progressive Rubella panencephalitis, rubella, and subacute sclerosing panencephalitis), fungal pneumonia (eg, histoplasmosis, coccidioidomycosis, blastosis, fungal infection in people with severely suppressed immune system (eg, Cryptococcus neoformans) Aspergillosis caused by Aspergillus spp .; Candidiasis caused by Candida; and Mucormycosis), Pneumocystis carinii (pneumocystis pneumonia), atypical pneumonia For example, Mycoplasma and Chlamydia spp.), Opportunistic pneumonia, nosocomial pneumonia, chemical pneumonia, and aspiration pneumonia (eg, pleurisy, pleural effusion, and pneumothorax (eg, simple spontaneous pneumothorax, complex spontaneous pneumonia) Pneumothorax, tension pneumothorax)), airway obstruction disease (eg asthma, chronic obstructive pulmonary disease (COPD), emphysema, acute bronchitis or chronic bronchitis), occupational lung disease (eg silicosis, black) Lung (coal miner pneumoconiosis), asbestosis, beryllium disease, occupational asthma, cotton pneumonia, and benign pneumoconiosis), invasive lung disease (eg pulmonary fibrosis (eg fibrosis alveolitis, normal type) Interstitial pneumonia), idiopathic pulmonary fibrosis, exfoliative interstitial pneumonia, lymphocytic interstitial pneumonia, unexplained histiocytosis (eg, Letterer-Give disease, hand-Schuller-Christian) Disease, eosinophilic granuloma), idiopathic pulmonary hemosiderin deposition, sarcoidosis and alveolar proteinosis), acute respiratory distress syndrome (eg also called adult respiratory distress syndrome), edema, pulmonary artery embolism, bronchitis (eg , Viral, bacterial), bronchiectasis, atelectasis, lung abscess (eg, caused by Staphylococcus aureus or Legionella pneumophila), and cystic fibrosis.

(Anti-angiogenic activity)
The naturally occurring equilibrium between the stimulatory factor and the inhibitor that is intrinsic to angiogenesis is the equilibrium in which the inhibitory effect predominates. Rastinejad et al., Cell 56: 345-355 (1989). In rare cases where neovascularization occurs under normal physiological conditions (eg, wound healing, organ regeneration, embryonic development, and female reproductive processes), angiogenesis is tightly regulated and spatial and temporal Determined. Under conditions of pathological angiogenesis (eg, characterizing solid tumor growth), control of these modulations is not possible. Unregulated angiogenesis becomes pathological and maintains the progression of many neoplastic and non-neoplastic diseases. Many serious diseases are dominated by abnormal neovascularization, including solid tumor growth and metastasis, arthritis, some types of eye disorders and psoriasis. See, for example, Moses et al., Biotech. 9: 630-634 (1991); Folkman et al. Engl. J. et al. Med. 333: 1757-1763 (1995); Auerbach et al., J. Biol. Microvasc. Res. 29: 401-411 (1985); Folkman, Advances in Cancer Research, edited by Klein and Weinhouse, Academic Press, New York, 175-203 (1985); Patz, Am. J. et al. Optalmol. 94: 715-743 (1982); and reviews by Folkman et al., Science 221: 719-725 (1983). In many pathological conditions, the process of angiogenesis contributes to the disease state. For example, significant data has been accumulated to suggest that solid tumor growth is dependent on angiogenesis. Folkman and Klagsbrun, Science 235: 442-447 (1987).

  The present invention provides for the treatment of diseases or disorders associated with neovascularization by administration of an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention. Malignant and metastatic conditions that can be treated with the polynucleotides and polypeptides of the invention, or agonists or antagonists, include the malignant diseases, solid tumors, and cancers described herein, as well as others known in the art. (For a review of such disorders, see, but not limited to, Fishman et al., Medicine, 2nd edition, JB Lippincott Co., Philadelphia (1985)). Accordingly, the present invention provides a method for the treatment of angiogenesis related diseases and / or disorders, which method encodes a therapeutically effective amount of an albumin fusion protein of the invention and / or an albumin fusion protein of the invention. Administering a polynucleotide to be treated to an individual in need thereof. For example, the fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention can be utilized in a variety of additional ways to therapeutically treat cancer or tumors. Cancers that can be treated with the fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention include prostate cancer, lung cancer, breast cancer, ovarian cancer, gastric cancer, pancreatic cancer, laryngeal cancer, esophageal cancer, and testicular cancer. Solid tumors, including liver cancer, parotid cancer, bile duct cancer, colon cancer, rectal cancer, cervical cancer, uterine cancer, endometrial cancer, kidney cancer, bladder cancer, thyroid cancer; primary tumor and metastasis; black Glioblastoma; capage sarcoma; leiomyosarcoma; non-small cell lung cancer; colorectal cancer; advanced malignant disease; and tumors arising from blood (eg, leukemia), but are not limited to these. For example, a fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention can be locally delivered to treat cancers such as skin cancer, head and neck tumors, breast tumors and Kaposi's sarcoma.

  In still other aspects, the fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention can be utilized to treat surface forms of bladder cancer, for example, by intravesical administration. The albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention can be delivered directly to or near the tumor site via injection or catheter. Of course, as the skilled artisan will appreciate, the appropriate mode of administration will vary with the cancer to be treated. Other modes of delivery are discussed herein.

  The albumin fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention may be useful in treating other disorders (including angiogenesis) in addition to cancer. These disorders include, but are not limited to: benign tumors (eg, hemangiomas, acoustic neuromas, neurofibromas, trachomas, and purulent granulomas); arteriosclerotic plaques; ocular angiogenesis Diseases (eg, diabetic retinopathy, immature retinopathy, macular degeneration, corneal transplant rejection, neovascular glaucoma, post-lens fiber proliferation, rubeosis, retinoblastoma, uveitis, and pterygium of the eye (Pterygia) (Abnormal vascular proliferation)); rheumatoid arthritis; psoriasis; delayed wound healing; endometriosis; angiogenesis; granulation; hyperplastic scar (keloid); pseudo-articular fracture; scleroderma; trachoma; Adhesion; Myocardial angiogenesis; Coronary collaterals; Cerebral collaterals; Arteriovenous malformation; Ischemic limb angiogenesis; Chromatography (Osler-Webber) syndrome; plaque neovascularization; telangiectasia; hemophilic joints; angiofibroma; fibromuscular dysplasia; wound granulation; Crohn's disease; and atherosclerosis.

  For example, in one aspect of the invention, hyperplastic scars comprising the step of administering to the hyperplastic scar or keloid an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention. A method is provided for treating keloids.

  In one embodiment of the invention, the fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is injected directly into a hyperplastic scar or keloid to prevent the progression of these lesions. The This treatment is particularly valuable in preventive treatment of conditions known to result in the development of hyperplastic scars and keloids (eg burns), and preferably the time during which the proliferative phase proceeds (the first injury) After about 14 days) but before the development of hyperplastic scars or keloids. As mentioned above, the present invention also provides a method for treating ocular neovascular diseases, including corneal neovascularization, neovascular glaucoma, proliferative diabetic retinopathy, post-lens fiber growth and macular degeneration. I will provide a.

  Further, ocular disorders associated with neovascularization that can be treated using the albumin fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to: : Angiogenic glaucoma, diabetic retinopathy, retinoblastoma, post lens fibroproliferation, uveitis, immature retinopathy, macular degeneration, corneal transplant neovascularization, and other ocular inflammatory diseases, ocular tumors, And diseases associated with neovascularization of the choroid or iris. For example, Waltman et al., Am. J. et al. Ophthal. 85: 704-710 (1978) and Gartner et al., Surv. Ophthal. 22: 291- 312 (1978).

  Accordingly, in one aspect of the invention, a therapeutically effective amount of a compound (albumin fusion protein of the invention and / or polynucleotide encoding the fusion protein of the invention) is administered to a patient such that blood vessel formation is inhibited. A method is provided for treating ocular neovascularization diseases, such as corneal neovascularization (including corneal transplant neovascularization), comprising administering to the cornea. Briefly, the cornea is a tissue that normally lacks blood vessels. However, in certain pathological conditions, capillaries can extend from the pericorneal vascular plexus to the cornea. When the cornea becomes vascularized, the cornea also becomes turbid, resulting in a loss of patient vision. When the cornea becomes completely opaque, vision loss can be complete. A wide variety of disorders can result, for example, in corneal neovascularization, including: corneal infections (eg, trachoma, herpes simplex keratitis, leishmaniasis and onchocercosis), immunological processes (eg, graft rejection) And Stevens-Johnson syndrome), alkaline burns, trauma, inflammation (of any cause), toxicity and nutritional deficiencies, and complications of wearing contact lenses.

  In particularly preferred embodiments, the present invention can be prepared for topical administration in saline (in combination with any preservatives and antibacterial agents commonly used in eye preparations) and eye drop forms Can be administered. Solutions or suspensions can be prepared in its pure form and can be administered several times a day. Alternatively, an anti-angiogenic composition prepared as described above can also be administered directly to the cornea. In a preferred embodiment, the anti-angiogenic composition is prepared with a mucoadhesive polymer that binds to the cornea. In a further embodiment, the anti-angiogenic factor or anti-angiogenic composition can be utilized as an adjunct to conventional steroid therapy. Topical treatment may also be useful prophylactically in corneal lesions that are known to have a high potential to induce angiogenic responses (eg, chemical burns). In these cases, treatment (possibly combined with steroids) can be started immediately to help prevent subsequent complications.

  In other embodiments, the compounds described above can be injected by an ophthalmologist under the guidance of a microscope directly into the corneal stroma. The preferred injection site may vary in the form of individual lesions, but the goal of administration is to place the composition on the advancing surface of the vasculature (ie, distributed between the blood vessels and the normal cornea). Is). In most cases, this involves a perilimbic corneal injection to “protect” the cornea from advancing blood vessels. This method can also be utilized immediately after corneal injury to prevent prophylactic corneal neovascularization. In this situation, the substance can be dispersed and injected into the perilimbic cornea between the corneal lesion and its unwanted potential blood supply edge. Such methods can also be utilized in a similar manner to prevent capillary infiltration of the transplanted cornea. In sustained release form, infusion may only be required 2-3 times a year. Steroids can also be added to the infusion solution to reduce inflammation resulting from the injection itself.

  In another aspect of the invention, administering to a patient a therapeutically effective amount of an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention so as to inhibit blood vessel formation. A method for treating neovascular glaucoma is provided. In one embodiment, the compound can be administered topically to the eye to treat an early form of neovascular glaucoma. In other embodiments, the compound can be implanted by injection into the area of the anterior chamber angle. In other embodiments, the compound can also be placed at any location so that the compound is continuously released into the aqueous humor. In another aspect of the invention, a patient is administered a therapeutically effective amount of an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention to the eye so that blood vessel formation is inhibited. A method for treating proliferative diabetic retinopathy comprising the steps is provided.

  In a particularly preferred embodiment of the invention, the proliferative diabetic retinopathy is an albumin fusion protein of the invention and / or a polynucleotide polynucleotide, polypeptide, antagonist and / or agonist encoding the albumin fusion protein of the invention in the retina. Can be treated by injection into the aqueous humor or vitreous to increase the local concentration of. Preferably, this treatment should be initiated before the acquisition of a serious disease that requires photocoagulation.

  In another aspect of the invention, a patient is administered a therapeutically effective amount of an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention to the eye so that blood vessel formation is inhibited. A method for treating post-lens fibroproliferation comprising the steps is provided. The compound can be administered locally via intravitreal injection and / or via intraocular implantation.

  In addition, disorders that can be treated with the fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to: hemangiomas, arthritis, psoriasis, vascular fibers Tumor, atheromatous plaque, delayed wound healing, granulation, hemophilic joint, hyperplastic scar, pseudoarticular fracture, Osler-Weber syndrome, pyogenic granulomas, scleroderma, trachoma, And vascular adhesion.

  Further, disorders and / or conditions that can be treated, prevented, diagnosed, and / or prognosticated with an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention include the following: Without being limited to: solid tumors, blood born tumors (eg, leukemia), tumor metastasis, capo sarcoma, benign tumors (eg, hemangiomas, acoustic neuromas, neurofibromas, trachomas, and purulent granulomas) ), Rheumatoid arthritis, psoriasis, ocular angiogenic diseases (eg, diabetic retinopathy, immature retinopathy, macular degeneration, corneal transplant rejection, neovascular glaucoma, post-lens fibroplasia, lebeosis, retinoblastoma, And uveitis), delayed wound healing, endometriosis, angiogenesis, granulation, hyperplastic scar (keloid) , Pseudo-articular fracture, scleroderma, trachoma, vascular adhesion, myocardial angiogenesis, coronary collaterals, cerebral collaterals, arteriovenous malformation, ischemic limb angiogenesis, Austler-Weber Syndrome, plaque neovascularization, telangiectasia, hemophilic joint, hemangiofibroma, fibromuscular dysplasia, wound granulation, Crohn's disease, atherosclerosis, birth control drugs (control menstruation, Such as by preventing angiogenesis necessary for embryo implantation, pathogenic consequences (eg, Roche minoria quintosa, ulcer (Helicobacter pylori), bartonellosis and bacterial hemangioma symptoms) With angiogenesis.

  In one aspect of the birth control method, the amount of the compound sufficient to block embryo implantation is administered before or after sexual intercourse and fertilization occurs, and thus an effective method of birth control, possibly “morning after. ) ”Method. The albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention can also be used in controlling menstruation or as a peritoneal lavage fluid in the treatment of endometriosis or for peritoneal transplantation Can be administered either for

  The albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention can be incorporated into surgical sutures to prevent stitch granulomas.

  The albumin fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention can be utilized in a wide variety of surgical procedures. For example, in one aspect of the invention, the composition (eg, in the form of a spray or film) separates normal surrounding tissue from malignant tissue and / or prevents the spread of disease to the surrounding tissue. To do so, it can be utilized to coat or spray the area prior to removal of the tumor. In other aspects of the invention, the composition (eg, in the form of a spray) is delivered via an endoscopic procedure to coat a tumor or to inhibit angiogenesis at a desired location. obtain. In still other aspects of the present invention, a surgical mesh coated with the anti-angiogenic composition of the present invention can be utilized in any procedure in which a surgical mesh can be utilized. For example, in one embodiment of the present invention, a surgical mesh raden with an anti-angiogenic composition provides support for the structure and releases a certain amount of anti-angiogenic factors. Therefore, it can be utilized during abdominal cancer resection surgery (eg, after colectomy).

  In a further aspect of the invention, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention after resection so that local recurrence of cancer and the formation of new blood vessels at the site are inhibited A method for treating a tumor resection site is provided, comprising administering to the resection margin of the tumor. In one embodiment of the invention, the anti-angiogenic compound is administered directly to the site of tumor resection (eg, by application, brushing, or otherwise using an anti-angiogenic compound to remove the tumor margin). Applied by coating). Alternatively, the anti-angiogenic compound can be incorporated into a known surgical paste prior to administration. In a particularly preferred embodiment of the invention, the anti-angiogenic compound is applied after hepatectomy and neurosurgery for malignancy.

  In one aspect of the invention, the fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention can be used in a wide variety of tumors, including breast tumors, colon tumors, brain tumors and liver tumors. Can be administered at the margin of resection. For example, in one embodiment of the invention, the anti-angiogenic compound can be administered to the site of a neurological tumor after resection so that the formation of new blood vessels at that site is inhibited.

  An albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention can also be administered with other anti-angiogenic factors. Representative examples of other anti-angiogenic factors include: anti-invasive factors, retinoic acid and its derivatives, paclitaxel, suramin, tissue inhibitors of metalloproteinase-1, tissue inhibitors of metalloproteinase-2, Plasminogen activation inhibitor-1, plasminogen activation inhibitor-2 and various forms of lighter “d group” transition metals.

  Lighter “d group” transition metals include vanadium, molybdenum, tungsten, titanium, niobium and tantalum species, for example. Such transition metal species can form transition metal complexes. Suitable complexes of the above transition metal species include oxo transition metal complexes.

  Representative examples of vanadium complexes include oxo vanadium complexes such as vanadate complexes and vanadyl complexes. Suitable vanadate complexes include, for example, metavanadate complexes and orthovanadate complexes such as ammonium metavanadate, sodium metavanadate and sodium orthovanadate. Suitable vanadyl complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate (including vanadyl sulfate hydrates such as vanadyl sulfate monohydrate and vanadyl sulfate trihydrate).

  Representative examples of tungsten and molybdenum complexes also include oxo complexes. Suitable oxo tungsten complexes include tungstate complexes and tungsten oxide complexes. Suitable tungstate complexes include ammonium tungstate, calcium tungstate, sodium tungstate dihydrate and tungstic acid. Suitable tungsten oxides include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxomolybdenum complexes include molybdate, molybdenum oxide and molybdenyl complexes. Suitable molybdate complexes include ammonium molybdate and its hydrate, sodium molybdate and its hydrate, and potassium molybdate and its hydrate. Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum (VI) oxide and molybdic acid. Suitable molybdenyl complexes include, for example, molybdenyl acetylacetonate. Other suitable tungsten and molybdenum complexes include, for example, hydroxo derivatives derived from glycerol, tartaric acid and sugars.

  A wide variety of other anti-angiogenic factors may also be utilized in the context of the present invention. Representative examples include: platelet factor 4; protamine sulfate; sulfated chitin derivatives (prepared from queen crab shell) (Murata et al., Cancer Res. 51: 22-26, 1991); sulfated polysaccharide peptidoglycan complex (SP-PG) (the function of this compound can be enhanced by the presence of steroids (eg, estrogen) and tamoxifen citrate); staurosporine; regulators of substrate metabolism ( Examples include proline analogs, cishydroxyproline, d, L-3,4-dehydroproline, thiaproline, α, α-dipyridyl, aminopropionitrile fumarate); 4-propyl-5- (4-pyridinyl)- 2 (3H) -oxazolone; methotrexate; mitozant Heparin; Interferon; 2 Macroglobulin-Serum; ChIMP-3 (Pavloff et al., J. Bio. Chem. 267: 17321-17326, 1992); Cyclodextrin tetradecasulfate; eponomycin; camptothecin; fumagillin (Ingber et al., Nature 348: 555-557, 1990); gold sodium thiomalate ("GST"; , 1987); anti-collagenase-serum; α2-antiplasmin (Holmes et al., J. Biol. Chem. 262 (4): 1659-1664, 1987). ); Bisantren (National Cancer Institute); disodium lobenzarit (N- (2) -carboxyphenyl-4-chloroanthronic acid, or “CCA”; Takeuchi et al., Agents Actions 36: 312- 316, 1992); thalidomide; Angostatic steroids; AGM-1470; carboxyaminoimidazole; and metalloproteinase inhibitors (eg, BB94).

(Disease at the cellular level)
Diseases associated with increased cell survival or inhibition of apoptosis that can be treated, prevented, diagnosed, and / or prognosticated by the fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention include cancer ( For example, follicular lymphoma, carcinoma with p53 mutation, and hormone-dependent tumor, these are: colon cancer, heart tumor, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, Includes testicular cancer, gastric cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelial tumor, osteoblastoma, giant cell tumor, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi sarcoma and ovarian cancer Autoimmune disorders (eg, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease (Be) cet's disease), Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) and viral infections (eg, herpesviruses, poxviruses and adenoviruses), inflammation, versus host grafts Disease, acute graft rejection, as well as chronic graft rejection.

  In a preferred embodiment, the fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is particularly for inhibiting cancer growth, progression and / or metastasis, as listed above. Used for.

  Additional diseases or conditions associated with increased cell survival that can be treated or detected by the fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention include malignant disease progression and / or metastasis and the following: Related disorders such as, but not limited to: leukemia (acute leukemia (eg, acute lymphocytic leukemia, acute myeloid leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic) And erythroleukemia) and chronic leukemia (eg, chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (eg, Hodgkin's disease and non-Hodgkin's disease), Multiple myeloma, Waldenstrom macroglobulinemia, heavy chain disease, and solid tumors (sarcomas and Carcinomas (eg, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphatic endothelial tumor, periosteum, mesothelioma, Ewing Tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell cancer, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, papillary adenocarcinoma , Cystadenocarcinoma, medullary cancer, primary bronchial cancer, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonic cancer, Wilms tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder Cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ventricular ependymoma, pineal gland, hemangioblastoma, acoustic neuroma, oligodendroglioma, Meningiomas, melanoma, neuroblastoma, Including fine retinoblastoma), but not limited to).

  Diseases associated with increased apoptosis that can be treated, prevented, diagnosed, and / or prognosticated by a fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention include, but are not limited to: Not limited to: AIDS; neurodegenerative disorders (eg, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration and brain tumors or previously related diseases); autoimmune disorders (eg, multiple Sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and chronic rheumatoid arthritis syndrome (Eg, aplastic anemia), versus host Graft disease, ischemic injury (such as caused by myocardial infarction, stroke and reperfusion injury), liver injury (eg, hepatitis-related liver injury, ischemia / reperfusion injury, cholestosis (bile duct injury) And liver cancer); toxic-induced liver disease (such as caused by alcohol), septic shock, cachexia and anorexia.

(Wound healing and epithelial cell proliferation)
According to a still further aspect of the invention, the fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention may be used for therapeutic purposes, eg for epithelial cell proliferation and basal keratinocytes for wound healing purposes. A process is provided that is utilized to stimulate and stimulate hair follicle formation and skin wound healing. An albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention may be clinically useful in stimulating wound healing, including: surgical wounds, resection wounds, deep wounds (Including dermal and epidermal damage), ocular tissue wounds, dental tissue wounds, oral wounds, diabetic ulcers, skin ulcers, elbow ulcers, arterial ulcers, venous stasis ulcers, heat exposure or chemistry Burns resulting from the substance, and other abnormal wound healing conditions (eg, uremia, malnutrition, vitamin deficiencies, and complications associated with steroids, radiation therapy and systemic treatment with antitumor drugs and antimetabolites. The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can be used after skin loss. It may be used to facilitate the peel of recovery.

  Albumin fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention increase adhesion of skin grafts to the wound bed and stimulate reepithelialization from the wound bed Can be used to The following are types of grafts in which an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention can be used to increase adhesion to a wound bed: autograft, artificial Skin, allograft, autograft, autoepidermic graft, avascular graft, Blair-Brown graft, bone graft, embryonic tissue graft, dermal graft, Delayed graft, skin graft, epidermal graft, fascia graft, full thickness skin graft, xenograft, xenograft, homologous graft, proliferative graft , Lamellar grafts, reticulated grafts, mucosal grafts, orier teal Grafts, omental grafts, patch grafts, stalk-like grafts, full thickness grafts, split skin grafts, thick split grafts . The albumin fusion protein of the present invention and / or a polynucleotide encoding the albumin fusion protein of the present invention can be used to promote skin strength and to improve the appearance of aged skin.

  The fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention are also believed to produce changes in hepatocyte proliferation and epithelial cell proliferation in the lung, breast, pancreas, stomach, small intestine, and large intestine. . The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention is an epithelial cell (for example, sebocyte, hair follicle, hepatocyte, type II alveolar epithelial cell). , Mucin producing goblet cells, and other epithelial cells, and their precursors contained within the skin, lung, liver, and gastrointestinal tract). The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can promote proliferation of endothelial cells, keratinocytes, and basal keratinocytes.

  The albumin fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention can also be used to reduce intestinal toxic side effects resulting from radiation, chemotherapy treatment or viral infection. The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention may have a cytoprotective effect on the small intestinal mucosa. The albumin fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention can also stimulate the healing of mucositis (mouth ulcers) resulting from chemotherapy and viral infection.

The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can be used for complete regeneration of the skin (ie, hair follicles) in full and partial thickness skin defects, including burns. Can be further used in the treatment of other skin defects such as psoriasis). Albumin fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention produce epidermolysis bullosa (causing frequent open and painful blisters by promoting re-epithelialization of these lesions, Deficiency of adhesion of the epidermis to the underlying dermis).
Albumin fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention also treat gastric and duodenal ulcers, and scar formation of the mucosal lining and glandular and duodenal mucosal It can be used to more quickly assist healing by regeneration of the inner layer. Inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis, are diseases that cause destruction of the mucosal surface of the small or large intestine, respectively. Accordingly, the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention promotes resurfacing of the mucosal surface to assist more rapid healing of inflammatory bowel disease. And can be used to prevent the progression of inflammatory bowel disease. Treatment with an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention is expected to have a significant effect on the production of mucus throughout the gastrointestinal tract and is ingested harmful It can be used to protect the intestinal mucosa from harmful substances, either from the substance or after surgery. The albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention can be used to treat diseases associated with its underexpression.

  Furthermore, the albumin fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention can be used to prevent and cure lung damage due to various pathological conditions. The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can increase the proliferation and differentiation of alveoli and brochiolar epithelium to prevent or treat acute or chronic lung injury. Can irritate and promote its repair. For example, emphysema resulting in progressive loss of alveoli, and inhalation injury resulting in necrosis of bronchiole epithelium and alveoli (ie resulting from smoke inhalation and burns) Polynucleotides or polypeptides, agonists or antagonists can be used to effectively treat. Also, albumin fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention can be used to stimulate the proliferation and differentiation of type II alveolar epithelial cells, which can be used in premature infants. It can help treat or prevent diseases such as pulmonary hyaline (eg, infant respiratory distress syndrome and bronchopulmonary dysplasia).

  Albumin fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention can stimulate hepatocyte proliferation and differentiation and thus liver diseases and conditions (eg, fulminant liver failure caused by cirrhosis) , Can be used to alleviate or treat viral hepatitis and toxic substances (ie, liver damage caused by acetaminophen, carbon tetrachloride, and other liver toxins known in the art).

  Furthermore, the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can be used to treat or prevent the development of diabetes mellitus. In a patient newly diagnosed with type I diabetes and type II diabetes, when some islet cell function remains, the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention It can be used to maintain its islet function so as to alleviate, delay or prevent the permanent manifestation of the disease. In addition, the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can be used as an aid in islet cell transplantation to improve or promote islet cell function.

(Neuron activity and neurological disorders)
The albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention can be used for diagnosis and / or treatment of diseases, disorders, injuries or injuries of the brain and / or nervous system. Nervous system diseases that can be treated with the compositions of the invention (eg, fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention) include axonal disconnection, neuronal loss or Examples include, but are not limited to, nervous system damage and diseases, disorders and / or conditions that cause either degeneration or demyelination. Nervous system lesions that can be treated in patients (including human patients and non-human mammalian patients) according to the present invention include any of the following central nervous system (including spinal cord, brain) or peripheral nervous system lesions: (1) Ischemic lesions (where oxygen deficiency in part of the nervous system results in neuronal damage or death, including cerebral infarction or ischemia, or spinal cord infarction or illness) (2) traumatic lesions (including lesions caused by physical injury or associated with surgery, including lesions that cut off parts of the nervous system, or compression lesions); (3) malignant lesions (here And a portion of the nervous system is destroyed or damaged by malignant tissue, which is either a nervous system related malignancy or a malignancy derived from non-neural tissue); (4) infectious lesions (where Part of the nervous system is destroyed or damaged, for example, as a result of infection by an abscess, or is associated with infection by human immunodeficiency virus, herpes zoster or herpes simplex virus, or associated with Lyme disease, tuberculosis, syphilis ); (5) Degenerative lesions, where a portion of the nervous system is destroyed or damaged as a result of the degenerative process, including Parkinson's disease, Alzheimer's disease, Huntington's disease or amyotrophic lateral sclerosis (ALS). (6) lesions associated with nutritional diseases, disorders and / or conditions (where a portion of the nervous system is destroyed by nutritional disorders or metabolic disorders). Or damaged, including vitamin B12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, mala (7) Neurological lesions associated with systemic disease (diabetes (diabetic neuropathy, bell palsy), systemic Including, but not limited to, systemic lupus erythematosus, cancer or sarcoidosis); (8) lesions caused by toxic substances (including alcohol, lead or certain neurotoxins); and (9) demyelinating lesions (here A portion of the nervous system is destroyed or damaged by demyelinating sovereignty, including multiple sclerosis, human immunodeficiency virus-related myelopathy, transverse myelopathy or various etiologies, progressive multifocal white matter brain Disorders, and bridge center myelin dissolution include, but are not limited to).

  In one embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used to protect neurons from the hypoxic damaging effects. In a further preferred embodiment, the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention is used for protecting nerve cells from the damaging action of cerebral hypoxia. According to this embodiment, the composition of the invention is used to treat or prevent neuronal cell damage associated with cerebral hypoxia. In one non-limiting aspect of this embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used to treat or prevent neuronal cell damage associated with cerebral ischemia. It is done. In another non-limiting aspect of this embodiment, the albumin fusion protein of the invention and / or a polynucleotide encoding the albumin fusion protein of the invention is used to treat or prevent neuronal cell damage associated with cerebral infarction. .

  In another preferred embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used to treat or prevent neuronal damage associated with stroke. In certain embodiments, the albumin fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention are used to treat or prevent cerebral nerve cell damage associated with stroke.

  In another preferred embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used to treat or prevent neuronal cell damage associated with a heart attack. In certain embodiments, albumin fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention are used to treat or prevent cerebral nerve cell damage associated with a heart attack.

The compositions of the invention useful for treating or preventing nervous system disorders can be selected by testing for biological activity in promoting neuronal survival or differentiation. For example, but not for purposes of limitation, compositions of the present invention that elicit any of the following effects may be useful according to the present invention: (1) in the presence or absence of hypoxia or hypoxia Increased survival time of neurons in either culture; (2) increased sprouting of neurons in medium or in vivo; (3) of neuron-related molecules (eg, choline acetyltransferase or acetylcholinesterase for motor neurons) in medium or in vivo. Increased production; or (4) reduced symptoms of neuronal dysfunction in vivo. Such an effect can be measured by any method known in the art.
Preferably, in a non-limiting embodiment, the increased survival time of the neuron is determined by methods described herein or otherwise known in the art (eg, Zhang et al., Proc Natl Acad Sci USA 97: 3637-42 (2000), or Arakawa et al., J. Neurosci. 10: 3507-3515 (1990)); increased sprouting of neurons is known in the art. Detected by known methods (eg, the method described in Pestronk et al. (Exp. Neurol. 70: 65-82 (1980)) or Brown et al. (Ann. Rev. Neurosci. 4: 17-42 (1981)). Obtained; increased production of neuron-related molecules can be achieved using techniques known in the art. And can be measured by bioassays, enzyme assays, antibody binding, Northern blot assays, etc. based on the molecule to be measured; and motor neuron dysfunction is a physical expression of motor neuron disorders (eg, weak Can be measured by assessing motor neuron conduction velocity, or dysfunction).

  In certain embodiments, motor neuron disorders that can be treated in accordance with the present invention include disorders that can affect motor neurons and other components of the nervous system (eg, infarcts, infections, exposure to toxins, trauma, surgical Injury, degenerative disease, or malignant disease), as well as disorders that selectively affect neurons (eg, amyotrophic lateral sclerosis) and include, but are not limited to, progressive spinal Muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, pediatric muscular atrophy and juvenile muscular atrophy, childhood progressive bulbar palsy (Fatio-Ronde disease), polio and post-polio symptoms, and hereditary movement Sensory neuropathy (Charcot-Marie-Tooth disease) is included, but is not limited to these.

  Furthermore, the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention may play a role in neuronal survival, synapse formation; transmission; Accordingly, the compositions of the present invention (including the fusion proteins of the present invention and / or the polynucleotides encoding the albumin fusion proteins of the present invention) have these roles, including but not limited to learning and / or recognition impairments. Can be used to diagnose and / or treat or prevent diseases or disorders associated with. The compositions of the present invention may be useful in the treatment or prevention of neurodegenerative disease states and / or behavioral disorders. Such neurodegenerative disease states and / or behavioral disorders include but are not limited to: Alzheimer's disease, Parkinson's disease, Huntington's disease, Tourette syndrome, schizophrenia, mania, dementia, paranoia, obsessive-compulsive disorder Disabilities, depression, panic disorder, learning disorders, ALS, psychosis, autism, and behavioral changes (including nutritional, sleep pattern, balance, and sensory disturbances). Furthermore, the compositions of the present invention may play a role in the treatment, prevention and / or detection of developing embryos, or developmental disorders associated with a sex disorder.

  Further, the fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention is from a disease, injury, disorder or injury associated with cerebrovascular disorders including but not limited to the following: May be useful in protecting nerve cells: cerebrovascular disorders (eg carotid artery thrombosis, carotid stenosis or moyamoya disease), amyloid angiopathy of the brain, cerebral aneurysm, cerebral hypoxia, cerebral arteriosclerosis Cerebral arteriovenous malformations, cerebral artery disease, cerebral embolism and thrombus (eg carotid artery thrombosis, sinus thrombosis and Wallenberg syndrome), epidural hematoma (eg epidural or subdural hematoma and Subarachnoid hemorrhage), cerebral fissure fracture, cerebral ischemia (eg, transient cerebral ischemia, subclavian artery steal syndrome, or vertebrobasis) ar insufficiency)), vascular dementia (e.g., multi-infarct dementia), periventricular leukomalacia, and vascular headache (e.g., cluster headache).

According to a still further aspect of the present invention, for therapeutic purposes, for example to stimulate neurological cell proliferation and / or differentiation, an albumin fusion protein of the invention and / or a polynucleotide encoding a fusion protein of the invention is used. A process is provided for use.
Thus, the albumin fusion proteins of the invention and / or polynucleotides encoding the fusion proteins of the invention can be used to treat and / or detect neurological diseases. Furthermore, the fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can be used as a marker or detection agent for a specific nervous system disease or injury.

  Examples of neurological diseases that can be treated or detected using a fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention include: brain disease (eg, maternal phenylketonuria) Cerebellar neoplasms including metabolic brain diseases including phenylketonuria such as infectious disease), pyruvate carboxylase deficiency, pyruvate dehydrogenase complex deficiency, Wernicke brain disorder, cerebral edema, infratentoral neoplasm Cerebellar ataxia, including spinal cord neoplasms, ventricular neoplasms such as choroid plexus neoplasms, hypothalamic neoplasms, supratent neoplasms, Canavan disease, spinocerebellar ataxia such as telangiectasia Cerebellar diseases such as cerebellar comotor disorders, Friedreich ataxia, Machado-Joseph disease Cerebral neoplasms such as olive bridge cerebellar atrophy, subtentorial neoplasms, diffuse periaxial encephalitis, spherical cell white matter atrophy, metachromatic leukotrophy, and subacute sclerosing panencephalitis Curing.

  Additional neurological diseases that can be treated or detected using the albumin fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention include the following: cerebrovascular disorders (eg, carotid artery thrombus) , Carotid artery disease including carotid artery stenosis and moyamoya disease), cerebral amyloid angiopathy, cerebral aneurysm, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebral artery disease, carotid artery thrombosis, sinus thrombosis and Wallenberg Cerebral embolism and thrombosis such as syndrome, cerebral hemorrhage such as epidural hematoma, subdural hematoma and subarachnoid hemorrhage, cerebral fissure fracture, transient cerebral ischemia, subclavian artery steal syndrome and vertebral brain Cerebral ischemia such as vertebrabasile insufficiency, vascular dementia such as multiple cerebral infarction dementia, brain Around leukomalacia, vascular headache such as cluster headache.

  Additional neurological diseases that can be treated or detected using albumin fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention include: dementia such as AIDS dementia complex, Presenile dementia such as Alzheimer's disease and Creutzfeldt-Jakob disease, senile dementia such as Alzheimer's disease and progressive supranuclear palsy, vascular dementia such as multiple cerebral infarction dementia, diffuse periaxial encephalitis Encephalitis, epidemic encephalitis, Japanese encephalitis, St. Louis encephalitis, tick-borne encephalitis and West Nile fever encephalitis, acute disseminated encephalomyelitis, uveal meningitis syndrome, post-encephalitis Parkinson's disease and subsphericity Meningoencephalomyelitis such as sclerosing panencephalitis, cerebral softening such as periventricular leukoplakia, systemic tendon including temporomandibular spasm Such as epilepsy, absence epilepsy, myoclonic epilepsy including MERRF syndrome, tonic / clonic epilepsy, complex partial epilepsy, partial epilepsy such as frontal lobe epilepsy and temporal lobe epilepsy, post-traumatic epilepsy, persistent partial epilepsy Status epilepticus, such as Harelfolden-Spatz syndrome.

  Additional neurological diseases that can be treated or detected using the fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention include the following: Dandy-Walker syndrome and normal pressure hydrocephalus Hydrocephalus, hypothalamic diseases such as hypothalamic neoplasms, cerebral malaria, narcolepsy including weakness attacks, bulbar poiomyelitis, cerebral pseudotumor, Rett syndrome, Reye syndrome, thalamic disease, cerebral toxoplasmosis , Intracranial tuberculoma and Zerweger syndrome, central nervous system infection such as AIDS dementia complex, brain abscess, subdural abscess, encephalomyelitis like equine encephalomyelitis, Venezuelan equine encephalomyelitis, necrosis Hemorrhagic encephalomyelitis, visna, and cerebral malaria.

  Additional neurological diseases that can be treated or detected with the fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention include the following: meningitis such as arachnoiditis, Aseptic meningitis such as viral meningitis including lymphocytic choriomeningitis, bacterial meningitis including Haemophilus meningitis, Listeria meningitis, Waterhouse-Friedersensen syndrome Meningococcal encephalomyelitis, pneumococcal meningitis and meningocerculosis, fungal meningitis like cryptococcal meningitis, subdural effusion, meninges like uveal meningoencephalitis syndrome Mioencephalitis, myelitis such as transverse myelitis, neurosyphilis such as spinal cord fistula, poliomyelitis including medullary polio and post-polio syndrome, prion disease For example, Creutzfeldt - Jakob disease, bovine spongiform encephalopathy, Gerusutokon - Sträussler syndrome, kuru, scrapie), and cerebral toxoplasmosis.

  Additional neurological diseases that can be treated or detected using the fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention include the following: Cerebellar neoplasms such as subtent neoplasms Includes central nervous system neoplasms such as brain neoplasms that contain living organisms, choroid plexus neoplasms, ventricular neoplasms such as hypothalamic neoplasms and epitenotic neoplasms, meningeal neoplasms, epidural neoplasms Spinal cord neoplasms, demyelinating diseases such as Canavan disease, diffuse cerebral sclerosis, including adrenal white matter dystrophy, diffuse periaxial encephalitis, spherical cell white matter atrophy, metachromatic leukodystrophy Diffuse cerebral sclerosis, allergic encephalomyelitis, necrotizing hemorrhagic encephalomyelitis, progressive multifocal leukoencephalopathy, multiple sclerosis, Hashinaka Spinal cord diseases such as central myelination, transverse myelitis, optic neuritis, scrapie, kyphosis, chronic fatigue syndrome, visna, high pressure nervous syndrome, meningopathy, congenital myotonia, Amyotrophic lateral sclerosis, spinal muscular atrophy such as Werdnich-Hoffmann disease, spinal cord compression, spinal cord neoplasm such as epidural neoplasm, syringomyelia, spinal cord fistula, stiff man syndrome, maternal 15q -13 Mental retardation such as microdefects, cat cry syndrome, de Lange syndrome, Down syndrome, gangliosidosis such as gangliosidosis G (M1), Zanthof disease, Tay-Sachs disease, Hartnap disease, homocystinuria Disease, Lawrence-Moon-Beedle syndrome, Lesch-Nyhan syndrome, maple syrup disease, flu Mucolipidosis such as saccharosis, neuronal ceroid sebaceous melanosis, ocular brain kidney syndrome, phenylketonuria such as maternal phenylketonuria, Prader-Villi syndrome, Rett syndrome, Rubinstein-Thebi syndrome, Tuberous sclerosis, WAGR syndrome, nervous system abnormalities such as total forebrain, neural tube defects such as incompetence, including water incompetence, Arnold-Chiari malformation, brain hernia, meningocele, meningocele Spinal fusion failure, such as cystic spina bifida and latent spina bifida.

  Additional neurological diseases that can be treated or detected using the fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention include the following: hereditary movements including Charcot-Marie disease And hereditary sensory neuropathy, hereditary visual atrophy, Lefsum disease, hereditary spastic paraplegia, Verdwig-Hoffmann disease, congenital analgesia and familial autonomic neuropathy and familial autonomic neuropathy, neuropathy Symptom manifestation (eg, agnosia including Gerstman syndrome), amnesia such as retrograde amnesia, apraxia, neurogenic bladder, weakness, hearing loss, partial hearing loss, loud recruit Communication disorders such as deafness, including mental disorders and tinnitus, aphasia, name aphasia, Broca aphasia and Language disorders such as aphasia, including Lunick's aphasia, dyslexia such as acute dyslexia, language developmental disorders, speech impairment such as aphasia, including aphasia, Broca's aphasia, and Vernicke's aphasia Communication disorders such as speech disorders, including speechlessness and stuttering, speech disorders such as aphasia and hoarseness, decerebral stiffness, delirium, fibrotic spasm, hallucinations, meningopathy, maternal 15 q- 13 Minor deficits, ataxia, athetosis, chorea, ataxia, hypoxia, hypotonia, movement disorders such as myoclonus, tics, torticollis and tremors, muscles such as muscle stiffness such as stiff man syndrome Nervous palsy such as facial palsy including hypertonia, muscle spasticity, ear shingles, gastric palsy, hemiplegia, ocular palsy such as double vision, Duane's syndrome, Horna Syndrome, chronic progressive extraocular palsy such as Keynes syndrome, bulbar palsy, tropical convulsions paraplegia, Brown-Sekar syndrome, limb paralysis, paraplegia such as respiratory paralysis and vocal cord paralysis, paralysis, phantom limbs , Taste disorders such as taste dysfunction and taste dysfunction, amblyopia, blindness, color blindness, double vision, semi-blindness, visual impairment such as visual dark spots and subnormal vision, Kleine-Levin syndrome, insomnia And sleep disorders such as hypersomnia including sleepwalking, spasticity such as opening disorders, coma, persistent plant condition and loss of consciousness such as fainting and dizziness, neuromuscular disorders such as congenital myotonia, Muscle atrophy such as amyotrophic lateral sclerosis, Lambert-Eaton myasthenia syndrome, motor neuropathy, spinal muscle atrophy, Charcot-Marie disease and Werdnich-Hoffmann disease Posterior polio syndrome, muscular dystrophy, myasthenia gravis, atrophic myotonia, congenital myotonia, nemarine myopathy, familial limb paralysis, multiple paramyloclonus, paired with tropical convulsions Paralysis and stiff man syndrome, peripheral nervous system diseases such as apical pain, renal amyloidosis, Ardy syndrome, Barre-Lieou syndrome, familial autonomic dysfunction, Horner syndrome, reflex sympathetic dystrophy and Shy-Drager syndrome Autonomic nervous system diseases such as: neurofibrosis 2 including cranial nerve diseases such as inner ear nerve diseases such as acoustic neuroma, facial nerve diseases such as facial neuralgia, Mercerson-Rosenthal syndrome, eye movements including amblyopia Sexual disorder, nystagmus, oculomotor paralysis, duane Ocular palsy such as symptom groups, chronic progressive extraocular palsy including Horner's syndrome, Keene's syndrome, strabismus such as esotropia and exotropia, oculomotor nerve palsy, ocular atrophy including hereditary ocular atrophy Ocular neurological diseases, optic disc crystal cavity, optic neuritis such as optic neuromyelitis, papillary edema, trigeminal neuralgia, vocal cord paralysis, demyelinating diseases such as optic myelitis and lordosis, such as diabetic foot Diabetic neuropathy.

  Additional neurological disorders that can be treated or detected using the fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention include the following: nerve squeezing syndromes such as carpal tunnel syndrome Thoracic outlet syndrome such as tarsal tunnel syndrome, cervical heel syndrome, ulnar nerve squeezing syndrome, causalgia, cervical neuralgia, neuralgia such as facial neuralgia and trigeminal neuralgia, experimental allergic neuritis, optic neuritis, polyneuropathy Inflammation, multiple radiculoneuritis and radiculitis (eg, multiple radiculitis, hereditary motor and sensory neuropathies (eg, Charcot-Marie disease, hereditary ocular atrophy, refsum disease, hereditary spastic paraplegia and Verdwig-Hoffmann disease, hereditary sensation and autonomic neuropathy (congenital analgesia and familiality) Law neurological include disorders), POEMS syndrome, sciatica, gustatory sweating syndrome and tetany)) neuritis like.

(Endocrine disorder)
Using the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention, treatment and prevention of disorders and / or diseases related to hormone imbalance and / or disorders or diseases of the endocrine system Diagnosis, and / or prognosis.

  Hormones secreted by the endocrine glands control physical development, sexual function, metabolism, and other functions. Disorders are categorized in two ways: disorders in hormone production and tissue failure in response to hormones. The pathogenesis of these hormonal imbalances and endocrine diseases, disorders or conditions can be genetic, somatic (eg, cancer and some autoimmune diseases), acquired (eg, by chemotherapy, injury or toxin), Or it can be infectious. Furthermore, the albumin fusion proteins of the invention and / or polynucleotides encoding the fusion proteins of the invention can be used as markers or detectors for specific diseases or disorders associated with endocrine systems and / or hormonal imbalances.

  Endocrine system and / or hormonal imbalance and / or disease includes disorders of uterine motility, including but not limited to: complications during pregnancy and delivery (eg, premature labor, late pregnancy) Spontaneous abortion, and delayed and stopped labor); and disorders and / or diseases of the menstrual cycle (eg, dysmenorrhea and endometriosis).

  Endocrine and / or hormonal imbalance disorders and / or diseases include pancreatic disorders and / or diseases (eg, diabetes mellitus, diabetes insipidus, congenital cataract defect, pheochromocytoma-islet cell tumor syndrome); Disorders and / or diseases (eg, Addison's disease, corticosteroid deficiency, masculinizing disease, hirsutism, Cushing syndrome, hyperaldosteronism, pheochromocytoma); pituitary disorders and / or diseases (eg, pituitary function) Hyperthyroidism, hypopituitarism, pituitary dwarfism, pituitary adenoma, panhypopituitarism, acromegaly, giantism); thyroid disorders and / or diseases (hyperthyroidism, thyroid function) Hypoxia, plumer disease, Graves disease (toxic scattered goiter), toxic nodular thyroid species, thyroiditis (Hashimoto thyroiditis, subacute granulomatous thyroiditis, and Silent lymphocytic thyroiditis), Pendred syndrome, myxedema, cretin disease, thyroid poisoning, thyroid hormone condensation disorder, thymine hypoplasia, thyroid Hurtle cell tumor, thyroid cancer, thyroid malignant tumor, medullary thyroid cancer Including, but not limited to); parathyroid disorders and / or diseases (eg, hyperparathyroidism, hypoparathyroidism); hypothalamic disorders and / or diseases.

  Furthermore, endocrine and / or hormonal imbalance disorders and / or diseases may also include testicular or ovarian disorders and / or diseases including cancer. Other testicular or ovarian disorders and / or diseases include, for example, ovarian cancer, polycystic ovary syndrome, Kleinfelter syndrome, burnishing testicular syndrome (bilateral testicular syndrome), Leydig cell birth defect, latent testicular disease, Noonan Further included are syndromes, myotonic dystrophy, testicular capillary species (benign), testicular neoplasia and new testis.

  In addition, endocrine system and / or hormonal imbalance disorders and / or diseases may also include, for example, polyendocrine hypofunction syndrome, pheochromocytoma, neuroblastoma, multiple endocrine adenomatosis, and endocrine tissue disorders and / or Can include cancer.

  In another embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is associated with a tissue in which the therapeutic protein corresponding to the therapeutic protein portion of the albumin fusion protein of the invention is expressed. Can be used to diagnose, prognose, prevent and / or treat endocrine diseases and / or disorders.

(Reproductive system disorders)
The albumin fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention can be used to diagnose, treat or prevent genital diseases and / or disorders. Genital disorders that can be treated by the compositions of the invention include genital damage, infections, neoplastic disorders, birth defects, and diseases or disorders that cause infertility, complications of pregnancy, labor or delivery, and postpartum However, it is not limited to these.

  Reproductive system disorders and / or diseases include testicular diseases and / or disorders, including: testicular atrophy, testicular gynecomastia, cryptic testis (unilateral and bilateral), testicular disease, ectopic Testis, epididymis and / or parotid testis (typically resulting from infection (eg, hemorrhoids, mumps, tuberculosis, and syphilis)), testicular torsion, nodular vasculitis, germ cell tumors (eg , Seminoma, fetal cell carcinoma, teratocarcinoma, choriocarcinoma, yolk sac tumor, teratoma), stromal tumor (eg Leydig cell adenoma), varicella, blood type, varicocele, semen aneurysm, inguinal Hernia and disorders of semen production (eg ciliary immobility syndrome, azoospermia, asthenozoospermia, azoospermia, hypospermia, teratospermia).

  Reproductive system disorders can also include, for example, prostate disorders such as: acute non-bacterial prostatitis, chronic non-bacterial prostatitis, acute bacterial prostatitis, chronic bacterial prostatitis, prostate cystitis, Prostate disease, granulomatous prostatitis, Malacoplatia, benign prostatic hyperplasia or thickening, and prostate neoplastic disorders (including adenocarcinoma), transitional cell carcinoma, ductal carcinoma, and squamous cell carcinoma.

  In addition, the compositions of the present invention are useful in the diagnosis, treatment and / or prevention of penile or urethral disorders or diseases including: inflammatory disorders (eg glans foreskinitis, obstructive dry balditis, phimosis) , Incarcerated phimosis, syphilis, herpes simplex virus, gonorrhea, non-gonococcal urethritis, chlamydia, mycoplasma, trichomonas, HIV, AIDS, Reiter's syndrome, acupuncture condyloma, flattened condyloma, and pearl penis papules); Premalignant lesions (including Koehler's erythema, Bowen's disease, Bowenoid baculosis, Bushke-Levenstein giant condyloma, and wart-like cancer); penile cancer (squamous cell carcinoma) , Including cancer in situ, wart-like cancer, and disseminated penile cancer); urethral neoplasia (urethra) Cotton carcinoma, bulbocavernosus urethral cancer, and prostatic urethra cancer); and erectile dysfunction (e.g., priapism, Peyronie's disease, erectile dysfunction, and impotence).

  Furthermore, vas deferens and / or disorders include vasculitis and CBAVD (congenital bilateral deficiency of the vas deferens); in addition, albumin fusion proteins of the invention and / or albumin fusion proteins of the invention The encoding polynucleotide can be used to diagnose, treat and / or prevent seminal vesicle diseases and / or disorders, including cystic diseases, congenital chloride diarrhea, and polycystic kidney disease.

  Other male reproductive tract disorders and / or diseases include, for example, Klinefelter syndrome, Young's syndrome, premature ejaculation, diabetes mellitus, cystic fibrosis, Cartagener syndrome, high fever, multiple sclerosis, and female breast .

  In addition, the polynucleotides, albumin fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention are used in the diagnosis, treatment and / or prevention of vaginal and vulvar diseases and / or disorders, including: Obtain: Bacterial vaginitis, Candida vaginitis, herpes simplex virus, soft lower vagina, inguinal granuloma, genopathic lymphogranuloma, scabies, human papilloma, vaginal trauma, vulva trauma, glandular disease, chlamydial vaginitis, hemorrhoids, hemorrhoids Trichomonas, acupuncture condylome, syphilis, infectious molluscum, atrophic vaginitis, Paget's disease, sclerotic atrophy, lichen planus, vulvar lesions, toxic shock syndrome, spasticity, vulvovaginitis, vulvar vestibularitis, and neoplasia Disorders (eg squamous hyperplasia, clear cell carcinoma of the kidney, basal cell carcinoma, melanoma, cancer of the Bartholin gland, and neovulvation Adult).

  Uterine disorders and / or diseases include: dysmenorrhea, retrograde uterus, endometriosis, fibroid, adenomyosis, anovulatory bleeding, amenorrhea, Cushing syndrome, hydatidiform mole, Scherman syndrome, early menopause, sexual prematurity, uterine polyps, dysfunctional malformed uterine bleeding (eg, due to vagal hormone signals), and neoplastic disorders (eg, adenocarcinoma, leiomyosarcoma, and sarcoma). Furthermore, the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention is useful as a marker or detector for uterine abnormalities as described below, and in the diagnosis, treatment and / or prevention of uterine abnormalities. Possible: Diagonal uterus, septum uterus, unilateral uterus, unilateral uterus with non-cavity failure angle, unilateral uterus with non-traffic cavity failure angle, unilateral uterus with traffic cavity angle, arch Uterus, two uterus cavities and a T-shaped uterus.

  Ovarian diseases and / or disorders include: anovulation, polycystic ovary (Stein-Levensall syndrome), ovarian cyst, ovarian dysfunction, ovary insensitive to gonadotropins, androgen ovarian excess Production, right ovarian vein syndrome, amenorrhea, hirsutism, and ovarian cancer (primary and secondary cancer growth, Sertoli-Leydig tumor, ovarian endometrioid cancer, ovarian papillary serous adenocarcinoma, ovarian mucinous adenocarcinoma, and ovarian crew Including, but not limited to, a Cambridge tumor).

  Cervical diseases and / or disorders include: cervicitis, chronic cervicitis, mucinous cervicitis, cervical dysplasia, cervical polyp, nerbot cyst, cervical erosion, cervix Dysfunction and cervical neoplasia (including, for example, cervical cancer, squamous cell, squamous cell carcinoma, adenosquamous cell neoplasia and columnar cell neoplasia).

  In addition, genital diseases and / or disorders include pregnancy disorders and / or diseases including: miscarriage and stillbirth (eg, early miscarriage, late miscarriage, spontaneous miscarriage, artificial miscarriage, therapeutic miscarriage, urgency) Miscarriage, abortion miscarriage, abortion miscarriage, complete miscarriage, habitual miscarriage, miscarriage miscarriage, and septic miscarriage); Disease, HELLP syndrome, normal placental early detachment, preplacental placenta, nausea, preeclampsia, eclampsia, gestational herpes, and pregnancy urticaria. Furthermore, the polynucleotides, poliopeptides, and agonists or antagonists of the present invention can be used to diagnose, treat and / or prevent diseases that can complicate pregnancy, including: heart disease, heart failure, rheumatic heart disease. , Congenital heart disease, mitral valve prolapse, hypertension, anemia, kidney disease, infection (eg rubella, cytomegalovirus, toxoplasmosis, infectious hepatitis, chlamydia, HIV, AIDS, and genital herpes), diabetes mellitus , Graves disease, thyroiditis, hypothyroidism, Hashimoto thyroiditis, chronic active hepatitis, cirrhosis, primary biliary cirrhosis, asthma, systemic lupus erythematosus, rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura Disease, appendicitis, ovarian cyst, gallbladder disorder, and intestinal obstruction.

  Complications related to parturition and childbirth include premature rupture of the membranes, preterm birth, post-term pregnancies, premature pregnancy, very slow progressing labor (laboratory prosthesis toy. ), Fetal asphyxia (eg, abnormal heart rate (fetal or maternal), respiratory problems, and abnormal fetal location), scapular dystocia, umbilical cord prolapse, amniotic fluid embolism, and abnormal uterine bleeding, It is done.

  In addition, postpartum diseases and / or disorders include endometritis, myometrium, parauterine connective tissue inflammation, peritonitis, pelvic thrombophlebitis, pulmonary embolism, endotoxemia, pyelonephritis, These include saphenous thrombophlebitis, mastitis, cystitis, postpartum hemorrhage, and inverted uterus.

  Other disorders and / or diseases of the female reproductive system that can be diagnosed, treated, and / or prevented by albumin fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention include, for example, Turner syndrome, Pseudo-Hemi-Yang Yang, Premenstrual Tension Syndrome, Pelvic Inflammatory Disease, Pelvic Congestion (Vascular Engagement), Insensitivity, Anorgasmia, Sexual Pain, Ruptured Fallopian Tube, Intermediate pain.

(Infectious diseases)
The albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention can be used to treat or detect infectious agents. For example, infectious diseases can be treated by raising the immune response, in particular by increasing proliferation and differentiation of B cells and / or T cells. The immune response can be raised by either raising an existing immune response or initiating a new immune response. Alternatively, the fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention can also directly inhibit infectious agents without necessarily eliciting an immune response.

  A virus is an example of an infectious agent that can cause a disease or condition that can be treated or detected by an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention. Examples of viruses include, but are not limited to, the following DNA and RNA viruses and viridae: arbovirus, adenoviridae, arenaviridae, arterivirus, birnaviridae, bunyaviridae, calcivirus Family, Sarcoviridae, Coronaviridae, Dengue fever, EBV, HIV, Flaviviridae, Hepadnaviridae (hepatitis), Herpesviridae (eg, cytomegalovirus, herpes simplex, herpes zoster), mono Negativevirus (eg, Paramyxoviridae, Measles virus, Rhabdoviridae), Orthomyxoviridae (eg, influenza A, influenza B, and parainfluenza), papillo Mavirus, Papovaviridae, Parvoviridae, Picornaviridae, Poxviridae (eg, Decubitus or Vaccinia), Reoviridae (eg, Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus) ), And Togaviridae (eg, genus Rubivirus). Viruses that fall within these families can cause a variety of diseases or conditions including but not limited to: arthritis, bronchiolitis, respiratory syncytium virus, encephalitis, ocular infections (eg, conjunctivitis) , Keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, chronic activity, delta), Japanese encephalitis B, Argentine hemorrhagic fever, Chingnia, Rift Valley fever, yellow fever, meninges Inflammation, opportunistic infection (eg AIDS), pneumonia, Burkitt lymphoma, chickenpox, hemorrhagic fever, measles, mumps, parainfluenza, rabies, cold, polio, leukemia, rubella, sexually transmitted disease, skin disease (Eg, capoji, warts), and viremia. Any of these symptoms or diseases can be treated or detected using the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention. In certain embodiments, a fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention is used for the following treatments: meningitis, dengue fever, EBV, and / or hepatitis (eg, Hepatitis B). In further specific embodiments, the fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention are used to treat patients who are non-responsive to one or more other commercially available hepatitis vaccines. The In a more specific embodiment, the fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used to treat AIDS.

  Similarly, bacterial or fungal factors that can cause disease or symptoms and that can be treated or detected by the albumin fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention include the following Gram negative And Gram-positive bacteria and bacteriophages and fungi, including but not limited to: Actinomyces (eg, Norcardia), Acinetobacter, Cryptococcus neoformans, Aspergillusis, Bacillaceae (eg, Bacillus anthracis) (eg, Bacillus anthracis) , Borrel a (e.g., Borrelia burgdorferi), Brucella, Candidia, Campyrobacter, Chlamydia, Clostridium (e.g., Clostridium botulinum, Clostridium dificile, Clostridium perfringes, Clostridium tetani) Coccidioidodes, Corynebacterium (e.g., Corynebacterium diptheriae), Cryptococcus, Dermatocycoses, E. coli (e.g., Enterotoxigenic E.coli and Enterohemorrhagic E.coli), Enterobacter (e.g., Enterobacter aerogenes), Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella typhi, Salmonella enteritidis, Salmonella typhi), Serratia, Yersinia, Shigella), Erysipelothrix, Hemophilus (eg, Hemophilus influenza B), Helicobacter, Legionella (eg, Legionella pneumophila) , Leptospira, Listeria (e.g., Listeria monocytogenes), Mycoplasma, Mycobacterium (e.g., Mycobacterium leprae and Mycobacterium tuberculosis), Vibrio (e.g., Vibrio cholerae), Neisseriaceae (e.g., Neisseriaceae gonorrhea, Neisseriaceae Meningitidis), Pasteurellacea, Proteus, Pseudomonas (e.g. , Pseudomonas aeruginosa), Rickettsiaceae, Spirochetes (eg, Treponema spp. Leptospira spp., Borrelia spp.), Shigella spp. Streptococcus (eg, Streptococcus aureus), Meningiococcus, Pneumococcus and Streptococcus (eg, Streptococcus pneumoniae and Group A, Groups B and C Streptococcus), and Ureaplas. These bacterial or fungal families can cause diseases or symptoms including but not limited to: antibiotic resistant infections, bacteremia, endocarditis, sepsis, eye infections (eg conjunctivitis), Uveitis, tuberculosis, gingivitis, bacterial diarrhea, opportunistic infections (eg infections associated with AIDS), peritonitis, prosthesis related infections, caries, Reiter's disease, respiratory tract infections (eg pertussis or Abscess), sepsis, Lyme disease, cat scratch disease, dysentery, paratyphoid fever, food poisoning, legionellosis, chronic inflammation and acute inflammation, erythema, yeast infection, typhoid fever, pneumonia, gonorrhea, meningitis (eg, meningitis type A) And B), chlamydia, syphilis, diphtheria, lei disease, brucellosis, peptic ulcer, anthrax, spontaneous abortion, birth defects, pneumonia, lung infection, hearing loss, blindness, somnolence, malaise, vomiting, chronic Diarrhea, Crohn's disease, colitis, hemorrhoid disease, infertility, pelvic inflammatory disease, candidiasis, paratuberculosis, tuberculosis, lupus, botulism, gangrene, tetanus, impetigo, rheumatic fever, scarlet fever, sexually transmitted disease, skin Diseases (eg cellulitis, dermatocycosis), venomemia, urinary tract infections, wound infections, noscomial infections. Any of these symptoms or diseases may be treated or detected using the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention. In certain embodiments, the albumin fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention are used to treat the following: tetanus, diphtheria, botulism, and / or type B Meningitis.

  Furthermore, diseases or symptoms caused by parasitic factors that can be treated, prevented and / or diagnosed by the fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention include the following families or classes: But not limited to: amebiasis, babesiosis, coccidiosis, cryptosporidiosis, dinucleamoebiasis, crossing, ectoparasites, giardia flagellate, helminthiasis, leishmaniasis, schistosomiasis (Sistisoma), Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas, and Sporozoan (eg, Plasmodium virax, Pla modium falciparium, Plasmodium malariae and Plasmodium ovale). These parasites can cause a variety of diseases or conditions including but not limited to: scabies, tsutsugamushi disease, ocular infections, intestinal diseases (eg, dysentery, Giardia flagellate disease), liver diseases, lungs Diseases, opportunistic infections (eg AIDS related), malaria, pregnancy complications, and toxoplasmosis. Any of these symptoms or diseases can be treated, prevented and / or diagnosed using the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention. In certain embodiments, the albumin fusion proteins of the invention and / or polynucleotides encoding the fusion proteins of the invention are used to treat, prevent and / or diagnose malaria.

  The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can be administered to a patient by administering an effective amount of the albumin fusion protein of the present invention or by removing cells from the patient. Either by supplying nucleotides to the cells and returning the manipulated cells to the patient (ex vivo treatment). Furthermore, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention may be used as an antigen in a vaccine to elicit an immune response against infectious diseases.

(Regeneration)
The albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention can be used to differentiate, proliferate and attract cells leading to tissue regeneration (Science 276: 59-87). (See 1997)). Using tissue regeneration, congenital defects, trauma (wounds, burns, incisions, or ulcers), aging, diseases (eg, osteoporosis, osteoarthritis, periodontal disease, liver failure), cosmetic plastic surgery May repair, replace, or protect tissue damaged by surgery, including fibrosis, reperfusion injury, or systemic cytokine damage.

  Tissues that can be regenerated using the present invention include: organs (eg, pancreas, liver, intestine, kidney, skin, endothelium), muscles (smooth muscle, skeletal muscle, or myocardium), vasculature (blood vessels). And lymphatic vessels), nerve, hematopoietic, and skeletal (bone, cartilage, tendon, and ligament) tissues. Preferably, regeneration occurs with no scarring or reduced scarring. Regeneration can also include angiogenesis.

  Furthermore, the fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention can increase the regeneration of tissues that are difficult to heal. For example, by increasing tendon / ligament regeneration, recovery time after injury is accelerated. The albumin fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention can also be used prophylactically in an attempt to avoid damage. Particular diseases that can be treated include tendinitis, carpal tunnel syndrome, and other tendon or ligament defects. Additional examples of tissue regeneration for non-healing wounds include pressure ulcers, vascular failure, surgical wounds, and ulcers associated with traumatic wounds.

  Similarly, nerve and brain tissue can also be regenerated by using a fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention to grow and differentiate nerve cells. Diseases that can be treated using this method include central and peripheral nervous system diseases, neurological disorders, or mechanical and traumatic disorders (eg, spinal cord disorders, head trauma, cerebrovascular diseases, and stroke ( stoke)). In particular, diseases associated with peripheral nerve injury, peripheral neuropathies (eg resulting from chemotherapy or other medical therapy), localized neuropathies, and central nervous system diseases (eg Alzheimer's disease, Parkinson's disease, Huntington) Disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome) are all treated, prevented and / or diagnosed using the albumin fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention. obtain.

(Gastrointestinal disorders)
The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention may be used to treat inflammatory diseases and / or conditions, infections, cancers (eg, intestinal neoplasms (carcinoid carcinomas of the small intestine, Non-Hodgkin lymphoma of the small intestine, lymphoma of the small intestine)), and gastrointestinal injuries including ulcers (eg, peptic ulcers) may be treated, prevented, diagnosed, and / or prognosticated.

  Gastrointestinal disorders include difficulty swallowing, swallowing pain, esophageal inflammation, peptic esophagitis, gastric reflux, gastric reflex, submucosal fibrosis and stenosis, Mallory-Weiss lesion, leiomyoma, lipoma, epidermoid carcinoma, gland Cancer, gastric remnant disorders, gastroenteritis, gastric atrophy, gastric / stomach cancer, gastric polyps, autoimmune diseases such as pernicious anemia, pyloric stenosis, gastritis (bacterial, virus) Sex, eosinophilic, stress-induced, chronic erosive, atrophic, plasma cells, and Menetrie disease), and peritoneal diseases (eg, chyle ascites, intraperitoneal hemorrhage, mesenteric sac, mesenteric lymphadenitis, intestinal tract) Membrane vascular occlusion, subcutaneous adipose histitis, neoplasm, peritonitis, pneumoperitoneum, infantile diaphragm abscess).

  Gastrointestinal disorders also include disorders related to the small intestine (eg, malabsorption syndrome, bloating, irritable bowel syndrome, glucose intolerance, abdominal disease, duodenal ulcer, duodenal inflammation, tropical sprue, whipple disease, Intestinal lymphangiectasia, Crohn's disease, appendicitis, ileal constipation, Meckel's diverticulum, multiple diverticula, complete rotation failure of the small and large intestines, lymphoma), and bacterial and parasitic diseases (eg travel Diarrhea, typhoid and paratyphoid fever, cholera, roundworms (Ascariassis lumbridices), worms (Ancylostoma duodenale), nematodes (Enterobius vermicularis), tapeworms (Taenia saginata) Echinococcus granulosus, Diphyllobothrium spp., And infection by T.Solium) and the like.

  Liver diseases and / or disorders include intrahepatic cholestasis (alagille syndrome, biliary cirrhosis), fatty liver (alcoholic fatty liver, Reye syndrome), hepatic vein thrombosis, hepatentricular degeneration, Hepatoma, hepatopulmonary syndrome, hepatorenal syndrome, portal hypertension (esophageal and gastric varices), liver abscess (amebic liver abscess), cirrhosis (alcoholic, bile and experimental), alcoholic liver disease ( Fatty liver, hepatitis, cirrhosis), parasitic (hepatic echinococcosis, cirrhosis, amoebic liver abscess), jaundice (hemolytic, hepatocellular, and cholestatic), cholestasis, portal pressure Hypertrophy, liver hypertrophy, ascites, hepatitis (alcoholic hepatitis, animal hepatitis, chronic hepatitis (autoimmunity, hepatitis B, hepatitis C, hepatitis D, drug-induced), toxicity Inflammation, viral human hepatitis (hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E), Wilson disease, granulomatous hepatitis, secondary biliary cirrhosis, hepatic Brain damage, portal hypertension, varicose veins, hepatic encephalopathy, primary biliary cirrhosis, primary sclerosing cholangitis, hepatocellular adenoma, hemangioma, bile stone, liver failure (hepatic) Brain disorders, acute liver failure), and liver neoplasms (angiomyolipoma, calcified liver metastasis, cystic liver metastasis, epithelial tumor, fibrolamellar hepatocarcinoma ), Focal nodular hyperplasia (focal nodular hy) perplasia), hepatocellular adenoma, hematologic biliary cystadenoma, germoma, hepatocellular carcinoma, hepatoma, liver cancer, hepatic hemangioendothelioma, mesenchymal hepatoma Mesenchymal tumor, nodular hyperplasia, benign liver cancer (hepatic cyst (Simple cyst), polycystic liver disease, hepatobiliary cystadenoma, common bile duct cyst, common bile duct cyst) (Mesenchymal hamartoma, infantile congenital hemangioendothelioma, hemangioma, liver purpura, lipoma, inflammatory pseudotumor, Miscellaneous), epithelial tumor (bile duct epithelium (bile) Hamartoma, bile duct adenoma), hepatocytes (adenoma, focal nodule hyperplasia, nodule hyperplasia)), metastatic liver tumor (hepatocytes, germoma, hepatocellular carcinoma, cholangiocellular, cholangiocarcinoma, cyst gland) Cancer, vascular cancer, hemangiosarcoma, capage sarcoma, hemangioendothelioma, other tumors, embryonic sarcoma, fibrosarcoma, leiomyosarcoma, rhabdomyosarcoma, carcinosarcoma, teratoma, carcinoid, squamous epithelium Cancer, primary lymphoma)), liver purpura, erythrohepatic porphyria, hepatic porphyria (acute intermittent porphyria, late cutaneous porphyria), Zellweger syndrome) .

  Pancreatic diseases and / or disorders include acute pancreatitis, chronic pancreatitis (acute necrotizing pancreatitis, alcoholic pancreatitis), neoplasms (pancreatic adenocarcinoma, cystadenocarcinoma, insulinoma, gastrinoma, and glucagonoma, cystic neoplasm Organisms, islet cell tumors, pancreatoblastoma, and other pancreatic diseases (eg, cystic fibrosis, cysts (pancreatic pseudocyst, pancreatic fistula, failure)).

  Gallbladder diseases include gallstones (cholelithiasis and common bile duct stones), post cholecystectomy syndrome, gallbladder diverticulosis, acute cholecystitis, chronic cholecystitis, bile duct tumors and lacrimal cysts.

  Colonic diseases and / or disorders include antibiotic-related colitis, diverticulitis, ulcerative colitis, acquired megacolon, abscess, fungal and bacterial infection, anorectal disease (eg fissure, sputum), Colon disease (colitis, colon neoplasm (colon cancer), adenomatous colon polyps (eg, choriocarcinoma), colon cancer, colorectal cancer), colonic diverticulitis, colonic diverticulum, giant colon ( Hirschsprung disease, harmful megacolon); sigmoid diseases (rectocolitis, sigmoin neoplasm)), constipation, Crohn's disease, diarrhea (infant diarrhea, dysentery), duodenal disease ( Duodenal neoplasms, duodenal obstruction, duodenal ulcer, duodenal inflammation, enteritis (enterocol is)), HIV enteropathy, ileal disease (ileal neoplasm, ileitis), immunoproliferative small bowel disease, inflammatory bowel disease (ulcerative colitis, Crohn's disease), intestinal obstruction, parasitic disease (anisakisis, baritidium) , Blastocystis infection, Cryptosporidium disease, Dinuclear amoebiasis, Amebic dysentery, Giardia flagellate disease, Intestinal fistula (rectal fistula), Intestinal neoplasm (blind neoplasm, colon neoplasm, Duodenal neoplasm, ileal neoplasm, intestinal polyp, jejunum neoplasm, rectal neoplasm), intestinal obstruction (imported leg syndrome, duodenal obstruction, impacted feces), intestinal pseudo-obstruction (cecal torsion) , Intussusception), intestinal perforation, intestinal Loop (colon polyp, Gardner syndrome, Poetz-Jegers syndrome), jejunum disease (jejunum neoplasm), malabsorption syndrome (blind snare syndrome, celiac disease), lactose intolerance, short bowel syndrome, tropical Sprue, whipple disease), mesenteric vascular occlusion, intestinal cystic emphysema, protein-losing enteropathy (intestinal lymphagiectasis), bowel disease (anal disease, fecal incontinence, Sputum, proctitis, intestinal fistula, ileum, rectal aneurysm), peptic ulcer (duodenal ulcer, peptic esophagitis, bleeding, perforation, gastric ulcer, Zollinger-Ellison syndrome), post gastrectomy syndrome (dumping syndrome) For example, hydrochloric acid deficiency, gastroduodenal reflux (bile reflux), Gastric fistula, gastric fistula obstruction, gastritis (atrophic or hypertrophic), gastric paralysis, gastric dilation, gastric diverticulum, gastric neoplasia (gastric cancer, gastric polyp) Gastric adenocarcinoma, proliferative gastric polyp), gastric rupture, gastric ulcer, gastric obstruction), tuberculosis, visceral hypoplasia, vomiting (e.g., blood loss, emesis of pregnancy, postoperative nausea and postoperative vomiting) and hemorrhagic colon A flame is mentioned.

  Further gastrointestinal diseases and / or disorders include bile duct disorders (eg gastric wall rupture), fistula (eg biliary fistula, esophageal fistula, gastric fistula, intestinal fistula, pancreatic fistula), neoplasms (eg new bile duct neoplasia) Organism, esophageal neoplasm (eg, adenocarcinoma of the esophagus), squamous cell carcinoma of the esophagus, gastrointestinal neoplasm, pancreatic neoplasm (eg, adenocarcinoma of the pancreas), mucinous cystic neoplasm of the pancreas, pancreatic cystic neoplasm, pancreas Blastoma and peritoneal neoplasms), esophageal diseases (eg, bullous disease, candidiasis, glycogen acantosis, ulceration, Barrett's esophageal varices, atresia, cysts, diverticulum (eg, Zenker's diverticulum), fistula (eg, trachea) Esophageal fistula), motility disorders (eg CREST syndrome, swallowing disorders, achalasia, spasticity, esophageal reflux), neoplasms, perforations (eg bourha Fe syndrome, Mallory-Weiss syndrome), stenosis, esophagitis, diaphragmatic hernia (eg hiatal hernia); gastrointestinal disease (eg gastroenteritis (eg cholera disease, Norwalk virus infection)), bleeding (eg vomiting, Melena, peptic ulcer bleeding), gastric neoplasm (gastric cancer, gastric polyp, gastric adenocarcinoma, gastric cancer)), hernia (eg congenital diaphragmatic hernia, femoral hernia, inguinal hernia, obturator hernia, umbilical hernia, abdominal wall hernia), And bowel disease (eg, blind-end disease (appendicitis, blind-end neoplasm)).

(Chemotaxis)
The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention may have chemotactic activity. Chemotactic molecules can direct cells (eg, monocytes, fibroblasts, neutrophils, T cells, mast cells, eosinophils, epithelial cells and / or endothelial cells) to specific sites in the body (eg, Attract or mobilize (sites of inflammation, infection, or hyperproliferation). The recruited cells can then repel and / or heal certain trauma or abnormalities.

  An albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention can increase the chemotactic activity of certain cells. These chemotactic molecules are then used to increase the number of cells targeted to a specific location in the body, thereby causing inflammation, infection, hyperproliferative disease, disorder and / or condition, or any Of the immune system. For example, chemotactic molecules can be used to treat wounds and other trauma to tissues by attracting immune cells to the injured location. The chemotactic molecules of the present invention can also attract fibroblasts, which can be used to treat wounds.

  It is also contemplated that the fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention may inhibit chemotactic activity. These molecules can also be used to treat disorders. Therefore, the fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can be used as a chemotaxis inhibitor.

(Binding activity)
The albumin fusion proteins of the invention can be used to screen for molecules that bind to the therapeutic protein portion of the fusion protein, or molecules that bind to the therapeutic protein portion of the fusion protein. Binding of the fusion protein to the molecule can activate (agonist), increase, inhibit (antagonist), or decrease the activity of the bound fusion protein or molecule. Examples of such molecules include antibodies, oligonucleotides, proteins (eg, receptors), or small molecules.

  Preferably, this molecule is closely related to a natural ligand (eg, a fragment of a ligand) of a therapeutic protein portion of a fusion protein of the invention, or a natural substrate, ligand, structural mimetic, or functional mimetic. (See Coligan et al., Current Protocols in Immunology 1 (2): Chapter 5 (1991)). Similarly, this molecule is closely related to the natural receptor to which the albumin fusion protein of the invention binds, or at least a fragment of the receptor that can be bound by the therapeutic protein portion of the albumin fusion protein of the invention (eg, the active site). Can do. In any case, the molecule can be rationally designed using known techniques.

  Preferably, screening for these molecules involves producing suitable cells that express the albumin fusion protein of the invention. Preferred cells include mammals, yeast, Drosophila, or E. coli. cells derived from E. coli.

  The assay may simply test the binding of the candidate compound to the albumin fusion protein of the invention, where the binding is detected by a label or in an assay for competition with a labeled competitor. In addition, the assay can test whether the candidate compound produces a signal produced by binding to the fusion protein.

  Alternatively, the assay can be performed using cell-free preparations, fusion proteins / molecules attached to a solid support, chemical libraries, or natural product mixtures. The assay also simplifies mixing candidate compounds with a solution containing an albumin fusion protein, measuring fusion protein / molecule activity or binding, and comparing fusion protein / molecule activity or binding to a standard. Can be included.

  Preferably, the ELISA assay may measure the level or activity of the fusion protein in a sample (eg, a biological sample) using a monoclonal or polyclonal antibody. The antibody may measure the level or activity of the fusion protein either by direct or indirect binding to the albumin fusion protein, or by competition with the albumin fusion protein for a substrate.

  Furthermore, the receptor to which the therapeutic protein portion of the albumin fusion protein of the present invention binds can be obtained in a number of ways known to those skilled in the art (eg, ligand panning and FACS sorting (Coligan et al., Current Protocols in Immun., 1 (2), No. 1). Chapter 5 (1991)) For example, expression cloning is used when polyadenylated RNA is prepared from cells that respond to this albumin fusion protein, eg, NIH3T3 cells (which are against FGF family proteins). SC-3 cells, and cDNA libraries made from this RNA, are divided into pools and COS cells that are not responsive to this albumin fusion protein. Or used to transfect other cells, transfected cells growing on glass slides are exposed to the albumin fusion protein of the invention after labeling them. Proteins can be labeled by a variety of means, including iodination or inclusion of recognition sites for site-specific protein kinases.

  After immobilization and incubation, the slides are subjected to autoradiographic analysis. Positive pools are identified and subpools are prepared and re-transfected using an iterative subpooling and rescreening process, ultimately resulting in a single clone encoding the putative receptor.

  As an alternative approach for receptor identification, labeled albumin fusion proteins can be linked to photomembrane or extract a preparation that expresses a receptor molecule for the therapeutic protein component of the albumin fusion protein of the invention. obtain. The crosslinked material is broken down by PAGE analysis and exposed to X-ray film. A labeled complex containing the receptor for the fusion protein can be excised, separated into peptide fragments, and subjected to protein microsequencing. Using the amino acid sequence obtained from microsequencing, a set of degenerate oligonucleotide probes is designed to screen a cDNA library and identify genes encoding putative receptors.

  Furthermore, using gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling techniques (collectively referred to as “DNA shuffling”), the fusion protein and / or the therapeutic protein portion of the albumin fusion protein of the present invention or The activity of the albumin component can be modulated, thereby effectively producing agonists and antagonists of the albumin fusion proteins of the present invention. In general, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, and Patten, P.M. A. Et al., Curr. Opinion Biotechnol. 8: 724-33 (1997); Harayama, S .; Trends Biotechnol. 16 (2): 76-82 (1998); Hansson, L .; O. Et al. Mol. Biol. 287: 265-76 (1999); and Lorenzo, M .; M.M. And Blasco, R .; See Biotechniques 24 (2): 308-13 (1998), each of these patents and publications incorporated herein by reference. In one embodiment, alteration of the polynucleotide encoding the albumin fusion protein of the invention can be achieved by DNA shuffling. DNA shuffling involves the construction of two or more DNA segments into a desired molecule by homologous recombination or site-specific recombination. In another embodiment, the polynucleotide encoding the albumin fusion protein of the invention, and the encoded albumin fusion protein may be subjected to error-prone PCR, random nucleotide insertion or other methods prior to recombination. Can be altered by subjecting to random mutagenesis. In another embodiment, one or more components, motifs, sections, portions, domains, fragments, etc. of the albumin fusion proteins of the invention are one or more components, motifs, sections, portions, one or more heterologous molecules. It can be recombined with domains, fragments and the like. In a preferred embodiment, the heterologous molecule is a family member. In a further preferred embodiment, the heterologous molecule is, for example, platelet derived growth factor (PDGF), insulin-like growth factor (IGF-I), transforming growth factor (TGF) -α, epidermal growth factor (EGF), fibroblasts. Cell growth factor (FGF), TGF-β, bone morphogenetic protein (BMP) -2, BMP-4, BMP-5, BMP-6, BMP-7, activin A and activin B, decapentaplegic (decapentaplegic) ( dpp), 60A, OP-2, dosarin, growth differentiation factor (GDF), nodal, MIS, inhibin-α, TGF-β1, TGF-β2, TGF-β3, TGF-β5, and nerves A growth factor such as glial-derived neurotrophic factor (GDNF).

  Other preferred fragments are biologically active fragments of the therapeutic protein portion and / or the albumin component of the albumin fusion proteins of the invention. Biologically active fragments exhibit activity similar to that of the therapeutic protein portion and / or albumin component of the albumin fusion protein of the invention, but not necessarily the therapeutic protein portion and / or albumin component of the albumin fusion protein of the invention. Fragments that are not identical to the activity of The biological activity of the fragment may include an improved desired activity or a reduced undesirable activity.

Furthermore, the present invention provides a method of screening a compound to identify a compound that modulates the action of the albumin fusion protein of the present invention. An example of such an assay involves combining mammalian fibroblasts, an albumin fusion protein of the invention, the compound to be screened and ³ [H] thymidine under cell culture conditions in which fibroblasts normally grow. To do. The control assay can be performed in the absence of the compound to be screened and the amount of ³ [H] thymidine incorporation in each case compared to the amount of fibroblast proliferation in the presence of this compound. By determination, it can be determined whether the compound stimulates proliferation. The amount of fibroblast proliferation is measured by liquid scintillation chromatography which measures the uptake of ³ [H] thymidine. Both agonist and antagonist compounds can be identified by this procedure.

  In another method, a mammalian cell or membrane preparation that expresses a receptor for the therapeutic protein component of the fusion protein of the invention is incubated with the labeled fusion protein of the invention in the presence of the compound. The ability of the compound to enhance or block the interaction can then be measured. Alternatively, a known second messenger system response and receptor according to the interaction of the compound to be screened is measured, and the ability of the compound to bind to the receptor and elicit a second messenger response is determined. Determine if it is a potential agonist or antagonist. Such second messenger systems include, but are not limited to, cAMP guanylate cyclase, ion channel or phosphoinositide hydrolysis.

  All of these above assays can be used as diagnostic or prognostic markers. Molecules discovered using these assays can be used to treat a disease or activate a patient with a specific outcome (eg, vascular proliferation) by activating or inhibiting a fusion protein / molecule. . Furthermore, the assay may discover factors that can inhibit or enhance the production of an albumin fusion protein of the invention from appropriately engineered cells or tissues.

  Accordingly, the invention includes a method of identifying a compound that binds to an albumin fusion protein of the invention comprising the following steps: (a) incubating the candidate binding compound with the albumin fusion protein of the invention; and (b ) Determining whether a bond has occurred; Furthermore, the present invention includes a method of identifying an agonist / antagonist comprising the following steps: (a) incubating a candidate compound with an albumin fusion protein of the present invention, (b) assaying biological activity. And (b) determining whether the biological activity of the fusion protein has been altered.

(Targeted delivery)
In another embodiment, the present invention provides a method of delivering a composition to a targeted cell that expresses a receptor for a component of an albumin fusion protein of the present invention.

  As discussed herein, the fusion proteins of the present invention may interact with heterologous polypeptides, heterologous nucleic acids, toxins or prodrugs via hydrophobic, hydrophilic, ionic and / or covalent interactions. Can meet. In one embodiment, the invention provides for the specific delivery of a composition of the invention to a cell by administering a fusion protein of the invention (including an antibody) associated with a heterologous polypeptide or nucleic acid. Provide a method. In one example, the present invention provides a method for delivering a therapeutic protein into a targeted cell. In another example, the invention can be a single-stranded nucleic acid (eg, antisense or ribozyme) or a double-stranded nucleic acid (eg, can be integrated into the genome of a cell, or can be replicated episomally and transcribed. , DNA) is provided for delivery to targeted cells.

  In another embodiment, the invention provides for the specific destruction of cells by administering an albumin fusion protein of the invention (eg, a polypeptide of the invention or an antibody of the invention) associated with a toxin or cytotoxic prodrug. Methods are provided for (eg, destruction of tumor cells).

  “Toxins” are endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or cells or cell surfaces that cause cell death under defined conditions Means a compound that binds and activates any molecule or enzyme not normally present. Toxins that can be used in accordance with the methods of the present invention include, but are not limited to: Radioisotopes known in the art, compounds that bind intrinsic or induced endogenous cytotoxic effector systems (Eg, antibody (or complement fixation including part thereof), thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonas endotoxin A, diphtheria toxin, saporin, momordin, gelonin) , Pokeweed antiviral protein, alpha-sarcin and cholera toxin, which is a non-toxic compound that is converted into a cytotoxic compound by enzymes normally present in the cell. Can be used in accordance with the method of the present invention. Cytotoxic prodrugs include, but are not limited to, glutamyl derivatives of benzoic acid mustard alkylating agents, phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside, daunorubicin, and phenoxyacetamide derivatives of doxorubicin. .

(Drug screening)
Use of the albumin fusion protein of the present invention or a polynucleotide encoding these fusion proteins for screening for an albumin fusion protein of the present invention or a molecule that alters the activity of a protein corresponding to the therapeutic protein portion of the albumin fusion protein. Further intended. Such methods include contacting the fusion protein with a selected compound suspected of having antagonist or agonist activity, and assaying the activity of the fusion protein following binding.

  The present invention is particularly useful for screening therapeutic compounds by using the albumin fusion proteins of the present invention, or binding fragments thereof, in any of a variety of drug screening techniques. Albumin fusion proteins used in such tests can be immobilized on a solid support, expressed on the cell surface, free in solution, or localized within the cell. One method of drug screening utilizes eukaryotic or prokaryotic host cells that are stably transformed with a recombinant nucleic acid expressing an albumin fusion protein. Drugs are screened against such transformed cells in competitive binding assays. For example, the formation of a complex between the agent being tested and the albumin fusion protein of the invention can be measured.

  Thus, the present invention provides a screening method for drugs or any other agent that affects the activity mediated by the albumin fusion protein of the present invention. These methods include contacting the agent with an albumin fusion protein of the invention or a fragment thereof by methods well known in the art, and of the complex between the agent and the albumin fusion protein or fragment thereof. Assaying for presence. In such competitive binding assays, the agent to be screened is typically labeled. After incubation, free drug is separated from the drug present in the bound form, and the amount of free or uncomplexed label is a measure of the ability of a particular drug to bind to the albumin fusion protein of the invention. is there.

  Another technique for drug screening provides high-throughput screening for compounds with appropriate binding affinity for the albumin fusion proteins of the present invention and European Patent Application 84/03564 (published September 13, 1984) (this) Are described in greater detail in US Pat. Briefly, a large number of different small peptide test compounds are synthesized on a solid substrate (eg, a plastic pin or some other surface). The peptide test compound is reacted with the albumin fusion protein of the invention and washed. The bound peptide is then detected by methods well known in the art. The purified albumin fusion protein is directly encoded on the plate for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.

  The present invention also contemplates the use of competitive drug screening assays, wherein neutralizing antibodies capable of binding the albumin fusion protein of the invention specifically compete with the test compound for binding to the albumin fusion protein or fragment thereof. . In this manner, the antibody is used to detect the presence of any peptide that shares one or more antigenic epitopes with the albumin fusion protein of the invention.

(Binding peptides and other molecules)
The present invention also includes screening methods for identifying polypeptides and non-polypeptides that bind to the albumin fusion proteins of the present invention, and binding molecules identified thereby. These binding molecules are useful, for example, as agonists and antagonists of the albumin fusion proteins of the present invention. Such agonists and antagonists may be used in accordance with the present invention in the therapeutic embodiments described in detail below.

This method includes the following steps:
a. Contacting the albumin fusion protein of the invention with multiple molecules; and b. Identifying a molecule that binds to the albumin fusion protein.

  The step of contacting the albumin fusion protein of the present invention with multiple molecules can be effected by a number of methods. For example, it may be contemplated that the albumin fusion protein is immobilized on a solid support and a solution of multiple molecules is contacted with the immobilized polypeptide. Such a procedure is similar to an affinity chromatography process using an affinity matrix composed of immobilized albumin fusion proteins of the present invention. Molecules with selective affinity for the albumin fusion protein can then be purified by affinity selection. The nature of the solid support, the process for attachment of the albumin fusion protein to this solid support, solvent, and conditions for affinity isolation or affinity selection are largely conventional and well known to those skilled in the art. .

  Alternatively, multiple polypeptides can also be separated into substantially separate fractions containing a subset of polypeptides or individual polypeptides. For example, a large number of polypeptides can be separated by methods known to those skilled in the art regarding separation of polypeptides such as gel electrophoresis, column chromatography and the like. Individual polypeptides can also be produced by transformed host cells (eg, recombinant phage) in such a way that they are expressed on or near the outer surface of the host cell. Individual isolates can then be “probed” by the albumin fusion protein, and between the albumin fusion protein and the individual clones in the presence of inducers that would be required for expression as needed. To determine if any selective affinity interaction has occurred. Prior to contacting the albumin fusion protein with each fraction containing individual polypeptides, these polypeptides can first be transferred to a solid support for further convenience. Such a solid support may simply be a piece of filter membrane made, for example, of nitrocellulose or nylon. In this manner, positive clones are identified from a population of expression libraries of transformed host cells that have a DNA construct that encodes a polypeptide that has a selective affinity for the albumin fusion protein of the invention. obtain. Furthermore, the amino acid sequence of a polypeptide having selective affinity for the albumin fusion protein of the invention can be determined directly by conventional means, or the coding sequence of the DNA encoding this polypeptide is often more convenient. Can be determined. The primary sequence can then be deduced from the corresponding DNA sequence. If the amino acid sequence is determined from the polypeptide itself, microsequencing techniques can be used. This sequencing technique can include mass spectrometry.

  In certain circumstances, it may be desirable to wash away unbound polypeptide from the albumin fusion protein of the invention and a mixture of multiple polypeptides before attempting to determine or detect the presence of a selective affinity interaction. possible. Such a washing step may be particularly desirable when the albumin fusion protein or multiple polypeptides of the present invention are bound to a solid support.

  A large number of molecules provided according to the method can be used as diversity libraries (eg, random or combinatorial peptide or non-peptide libraries that can screen for molecules that specifically bind to the albumin fusion proteins of the invention). Can be provided. Many libraries that can be used are known in the art, such as chemical synthesis libraries, recombinant libraries (eg, phage display libraries), and in vitro translation-based libraries. Examples of chemical synthesis libraries are described below: Fodor et al., 1991, Science 251: 767-773; Houghten et al., 1991, Nature 354: 84-86; Lam et al., 1991, Nature 354: 82-84; Medynski, 1994, Bio / Technology 12: 709-710; Gallop et al., 1994, J. Biol. Medicinal Chemistry 37 (9): 1233-1251; Ohlmyer et al., 1993, Proc. Natl. Acad. Sci. USA 90: 10922-10926; Erb et al., 1994, Proc. Natl. Acad. Sci. USA 91: 11142-11426; Houghten et al., 1992, Biotechniques 13: 412; Jayawickreme et al., 1994, Proc. Natl. Acad. Sci. USA 91: 1614-1618; Salmon et al., 1993, Proc. Natl. Acad. Sci. USA 90: 11708-11712; PCT Publication No. WO93 / 20242; and Brenner and Lerner, 1992, Proc. Natl. Acad. Sci. USA 89: 5381-5383.

  Examples of phage display libraries are described below: Scott and Smith, 1990, Science 249: 386-390; Devlin et al., 1990, Science, 249: 404-406; Christian, R .; B. Et al., 1992, J. Am. Mol. Biol. 227: 711-718); Lenstra, 1992, J. MoI. Immunol. Meth. 152: 149-157; Kay et al., 1993, Gene 128: 59-65; and PCT Publication No. WO 94/18318 (August 18, 1994).

  In vitro translation-based libraries include: PCT Publication No. WO91 / 05058 (April 18, 1991); and Mattheakis et al., 1994, Proc. Natl. Acad. Sci. USA 91: 9022-9026, but are not limited to these.

  As an example of a non-peptide library, a benzodiazepine library (see, eg, Bunin et al., 1994, Proc. Natl. Acad. Sci. USA 91: 4708-4712) can be adapted for use. A peptoid library (Simon et al., 1992, Proc. Natl. Acad. Sci. USA 89: 9367-9371) can also be used. Another example of a library that can be used is described by Ostresh et al. (1994, Proc. Natl. Acad. Sci. USA 91: 11138-11142), where the amide functionality in the peptide is over-methylated. (Permethylated) to produce a chemically transformed combinatorial library.

  The various non-peptide libraries that are useful in the present invention are large. For example, Ecker and Crooke (1995, Bio / Technology 13: 351-360) include benzodiazepines, hydantoins, piperazinediones, biphenyls, sugar analogs, β-mercaptoketones among the species that form the basis of various libraries. , Arylacetic acid, acylpiperazine, benzopyran, cubane, xanthine, amine imide and oxazolone.

  Non-peptide libraries can be broadly classified into two types: modified monomers and oligomers. The modified monomer library uses a relatively simple skeletal structure on which various functional groups are added. Often the scaffold is a molecule with known useful pharmacological activity. For example, the skeleton can be a benzodiazepine structure.

  Non-peptide oligomer libraries use a large number of monomers that are assembled together in a manner that creates a new shape depending on the order of the monomers. Among the monomer units used are carbamates, pyrrolinones and morpholinos. Peptoids (peptide-like oligomers whose side chains are attached to the alpha amino group rather than the alpha carbon) form the basis of another version of a non-peptide oligomer library. The first non-peptide oligomer library uses a single type of monomer and thus contains a repetitive backbone. Modern libraries use more than one monomer and give a library with added degrees of freedom.

  Screening the library can be accomplished by any of a variety of commonly known methods. See, for example, the following references disclosing screening of peptide libraries: Parmley and Smith, 1989, Adv. Exp. Med. Biol. 251: 215-218; Scott and Smith, 1990, Science 249: 386-390; Fowlkes et al., 1992; BioTechniques 13: 422-427; Oldenburg et al., 1992, Proc. Natl. Acad. Sci. USA 89: 5393-5397; Yu et al., 1994, Cell 76: 933-945; Staudt et al., 1988, Science 241: 577-580; Bock et al., 1992, Nature 355: 564-566; Tuerk et al., 1992, Proc. Natl. Acad. Sci. USA 89: 6988-6992; Ellington et al., 1992, Nature 355: 850-852; US Pat. No. 5,096,815, US Pat. No. 5,223,409, and US Pat. No. 5,198,346 ( All to Ladner et al.); Rebar and Pabo, 1993, Science 263: 671-673; and PCT Publication No. WO94 / 18318.

  In certain embodiments, screening to identify molecules that bind an albumin fusion protein of the invention comprises contacting a member of the library with an albumin fusion protein of the invention immobilized on a solid phase, and albumin fusion protein Can be carried out by collecting those library members that bind to. An example of such a screening method, referred to as “panning”, is by way of example Palmley and Smith, 1988, Gene 73: 305-318; Fowles et al., 1992, BioTechniques 13: 422-427; PCT Publication No. WO94 / 427. 18318; as well as the references cited therein.

  In another embodiment, a two-hybrid system for selecting interacting proteins in yeast (Fields and Song, 1989, Nature 340: 245-246; Chien et al., 19991, Proc. Natl. Acad. Sci. USA 88: 9578-9582) can be used to identify molecules that specifically bind to the polypeptides of the invention.

  When the binding molecule is a polypeptide, the polypeptide can be conveniently selected from any peptide library (including random peptide libraries, combinatorial peptide libraries, or bias peptide libraries). The term “biased” means that, in the peptide in this case, the method of generating the library is manipulated to limit one or more parameters that govern the diversity of molecular collection that occurs. As used herein.

  Thus, a truly random peptide library creates a collection of peptides where the probability of finding a particular amino acid at a given position in the peptide is the same for all 20 amino acids. However, for example, by identifying that lysine occurs every 5 amino acids or by pinning and specifying that the 4th, 8th and 9th positions of the decapeptide library contain only arginine, the bias is Can be introduced. Obviously, many types of bias can be contemplated and the invention is not limited to any particular bias. Furthermore, the present invention contemplates specific types of peptide libraries (eg, libraries that use a phage display peptide library and a DNA construct comprising a lambda phage vector with a DNA insert).

  As noted above, in the case of a binding molecule (which is a polypeptide), the polypeptide has from about 6 to less than about 60 amino acid residues, preferably from about 6 to about 10 amino acid residues, and more preferably It can be about 6 to about 22 amino acids. In another embodiment, the binding polypeptide has in the range of 15-100 amino acids, or in the range of 20-50 amino acids.

  The selected binding polypeptide can be obtained by chemical synthesis or recombinant expression.

(Other activities)
The albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention can be used in various disease states (eg, thrombosis, arteriosclerosis, and other) as a result of the ability to stimulate vascular endothelial cell proliferation. It can be utilized in procedures for stimulating revascularization of ischemic tissue due to cardiovascular conditions). An albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention may also be utilized to stimulate angiogenesis and limb remodeling as discussed above.

  The albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention may also be utilized for the treatment of wounds resulting from injury, burns, post-operative tissue repair, and ulcers. Because they are mitogenic to various cells of different origin (eg, fibroblasts and skeletal muscle cells) and thus promote repair or replacement of damaged or diseased tissue.

  An albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention also stimulates neuronal growth and can be associated with certain neuronal disorders or neurodegenerative conditions (eg, Alzheimer's disease, Parkinson's disease, and It can be used to treat and prevent neuronal damage that occurs in AIDS-related complexes). The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention may have the ability to stimulate chondrocyte proliferation, and thus enhance bone and periodontal remodeling. And can be utilized for assistance in tissue grafts or bone grafts.

  The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can also be utilized to prevent skin aging due to sunburn by stimulating keratinocyte proliferation.

  The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention may also be utilized to prevent hair loss. This is because members of the FGF family activate hair-forming cells and promote melanocyte proliferation. Proliferation and differentiation of hematopoietic and bone marrow cells when used in combination with other cytokines utilizing the albumin fusion protein of the present invention and / or polynucleotide encoding the albumin fusion protein of the present invention along the same series Can irritate.

  Albumin fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention can also be utilized to maintain organs prior to transplantation or can be used to support primary tissue cell culture. . The albumin fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention can also be utilized to induce tissue of mesodermal origin for differentiation in early embryos.

  The albumin fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention can also increase or decrease embryonic stem cell differentiation or proliferation in addition to the hematopoietic lineage as discussed above.

  The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention may also have mammalian characteristics (eg, height, weight, hair color, eye color, skin, adipose tissue proportion, pigment Can be used to adjust deposition, size, and shape (eg, cosmetic surgery). Similarly, albumin fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention modulate mammalian metabolism affecting catabolism, anabolism, processing, utilization, and energy storage. Can be used for.

  The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention is biorhythm, caricadic rhythm, depression (including depressive disorder), tendency of violence, resistance to pain, By affecting fertility (preferably by activin or inhibin-like activity), hormone or endocrine levels, appetite, libido, memory, stress, or other cognitive quality, Can be used to change.

  Albumin fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention also increase storage capacity, fat content, lipids, proteins, carbohydrates, vitamins, minerals, cofactors, or other nutritional components Or it can be used as a food additive or preservative to reduce.

  The applications cited above have uses in a wide variety of hosts. Such hosts include humans, mice, rabbits, goats, guinea pigs, camels, horses, mice, rats, hamsters, pigs, micro-pigs, chickens, goats, cows, sheep, dogs, cats, non-humans. This includes, but is not limited to, primates and humans. In certain embodiments, the host is a mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig, sheep, dog or cat. In preferred embodiments, the host is a mammal. In the most preferred embodiment, the host is a human.

  As generally described the present invention, the same is readily understood and provided as indicated in the following examples and is not intended to be limiting.

  Without having further explanation, those skilled in the art will be able to make and utilize the variations found in the present invention using the foregoing description and the following example instructions and implement the methods as claimed. obtain. Accordingly, the following examples are not constructed to specifically indicate preferred embodiments of the invention and to limit the remainder of the disclosure in any way.

Example 1: Generation of pScNHSA and pScCHSA Vectors pScNHSA (ATCC Trust Number PTA-3279) and pScCHSA (ATCC Trust Number PTA-3276) pPPC0005 (ATCC Trust Number PTA-3278) derivatives and therapeutic proteins or fragments Alternatively, a polynucleotide encoding a variant thereof is used as a cloning vector inserted adjacent to or into the translation frame having a polynucleotide encoding human serum albumin “HSA.” PScCHSA is a therapeutic vector. PScNHSA can be used to generate HSA therapeutic protein fusions, while it can be used to generate industrial protein HSA fusions.

(Production of pScCHSA: Albumin fusion with the C-terminus of the albumin moiety to the therapeutic moiety)
A vector for facilitating cloning of the DNA encoding the therapeutic protein N-terminus relative to the DNA encoding mature albumin protein is modified so that the nucleic acid sequence encoding the chimeric HSA signal peptide in pPPC0005 contains an XhoI site and a ClaI site. It was prepared by.

  First, the XhoI and ClaI sites unique to pPPC0005 (located 3 ′ of the ADH1 termination sequence) are eliminated by digesting pPPC0005 with XhoI and ClaI, and the blunt ends are religated with T4 DNA polymerase. Thus, pPPC0006 was produced.

Next, the XhoI restriction enzyme recognition site and the ClaI restriction enzyme recognition site encode the signal peptide of HSA (kex2 site from HSA leader chimera and conjugation factor α “MAF”) in pPPC0006 using 2-round PCR. Engineered in the nucleic acid sequence.
In the first round of PCR, amplification was performed using the primers shown as SEQ ID NO: 1039 and SEQ ID NO: 1040. The primer set forth as SEQ ID NO: 1039 includes a nucleic acid sequence encoding a portion of the HSA signal peptide sequence, a kex2 site from the mating factor alpha leader sequence, and the amino terminal portion of the mature form of HSA. Four point mutations were introduced into this sequence to create the XhoI and ClaI sites found at the junction of the chimeric signal peptide and the mature form of HSA. These four point mutations are shown underlined in the sequence shown below. In pPPC0005, the nucleotides at these four positions from 5 ′ to 3 ′ are T, G, T and G.

5'-GC C T C GA G AAAAGAGATGCACACAAGAGTGAGGTTGCTCATCG A TTTAAAGATTTGGG-3 '( SEQ ID NO: 1039) and 5'-AATCGATGAGCAACCTCACTCTTGTGTGCATCTCTTTT
CTCGAGGCTCTCTGGAATAAGC-3 ′ (SEQ ID NO: 1040). A second round of PCR was then performed using upstream flanking primer 5′-TACAAACTTAAGAGTCCAATTTAGC-3 ′ (SEQ ID NO: 1041) and downstream flanking primer 5′-CACTTCTCTAGAGTGGTTTCATATGTTCTT-3 ′ (SEQ ID NO: 1042). The resulting PCR product was then purified and digested with Afl II and Xba I and ligated to the same site in pPPC0006 to create pScCHSA. The resulting plasmid has Xho I and Cla I sites engineered into signal sequences. The presence of the Xho I site results in a one amino acid change at the end of the signal sequence from LDKR to LEKR. This D to E change involves a polynucleotide encoding the therapeutic moiety of an albumin fusion protein having a 5 ′ Sal I site (which is compatible with the Xho I site) and a 3 ′ Cla I site. If the nucleic acid sequence is linked to the Xho I and Cla I sites of pScCHSA, it is not present in the final albumin fusion protein expression plasmid. The ligation of Sal I and Xho I restores the original amino acid sequence of the signal peptide sequence. DNA encoding the therapeutic moiety of the albumin fusion protein can be inserted after the Kex2 site (Kex2 cuts after the last dibasic amino acid sequence KR of the signal peptide) and before the ClaI site.

(Preparation of pScNHSA: albumin fusion of albumin part N-terminal to therapeutic part)
A vector that facilitates cloning of the DNA encoding the therapeutic protein portion into the C-terminus of the DNA encoding mature albumin protein was created by adding three 8-base pair restriction sites to pScCHSA. Asc I, Fse I, and Pme I restriction sites were added between the Bsu36 I and Hind III sites at the end of the nucleic acid sequence encoding the mature HSA protein. This was achieved by the use of two complementary synthetic primers (SEQ ID NO: 1043 and SEQ ID NO: 1044) containing the underlined Asc I restriction site, Fse I restriction site, and Pme I restriction site.

5′-AAGCTGCCTTAGGCTTATAATAA GGCGCGGCCGGCCGGCCGTTTAAAC TAAGCTTAATTCT-3 ′ (SEQ ID NO: 1043) and 5-AGATATAAGCTTA GTTTAAAACGGCCGGCCGGCGGCCCTTTATATAGTCTAAGGCAGCT4 . These primers were annealed with Bsu36 I and Hind III and digested and bound to similar sites in pScCHSA creating pScNHSA.

Example 2: Generation of a general construct for yeast transformation
The vectors pScNHSA and pScCHSA can be used as cloning vectors in which a polynucleotide encoding a therapeutic protein or fragment or variant thereof is inserted adjacent to a polynucleotide encoding mature human serum albumin “HSA”. While pScCHSA is used to generate therapeutic fusion HSA fusions, pScNHSA can be used to generate HSA therapeutic protein fusions.

(Production of albumin fusion constructs containing HSA-therapeutic protein fusion products)
DNA encoding the therapeutic protein (eg, the sequence shown in SEQ ID NO: X or known in the art) can be used to create a seamless fusion by adding primers (eg, restriction sites) that facilitate the production of the fusion construct. Can be amplified by PCR using, for example, by encoding a linker sequence. For example, one of ordinary skill in the art will recognize a 5 ′ primer that adds a polynucleotide encoding the last four amino acids of the mature form of HSA on the 5 ′ end of the DNA encoding the therapeutic protein (and including the Bsu36I site); and a stop codon and A 3 ′ primer can be designed that adds an appropriate cloning site on the 3 ′ end of the therapeutic protein encoding sequence. For example, the forward primer used to encode the therapeutic protein is a sequence

（配列番号１０４５）を有し得、ここで下線が引かれた配列は、Ｂｓｕ３６Ｉ部位であり、大文字のヌクレオチドは、成熟ＨＳＡタンパク質の最後の４つのアミノ酸（ＡＬＧＬ）をコードし、そして（Ｎ）_１５は、目的の治療タンパク質をコードする最初の１５のヌクレオチドと同一である。同様に、治療タンパク質をコードするＤＮＡを増幅するのに用いられる逆方向プライマーは、配列

(SEQ ID NO: 1045), where the underlined sequence is the Bsu36I site, the uppercase nucleotide encodes the last four amino acids (ALGL) of the mature HSA protein, and (N) ₁₅ is identical to the first 15 nucleotides encoding the therapeutic protein of interest. Similarly, the reverse primer used to amplify the DNA encoding the therapeutic protein is the sequence

（配列番号１０４６）を有し得、ここでイタリック体で書かれた配列は、Ｐｍｅ Ｉ部位であり、二重に下線が引かれた配列は、Ｆｓｅ Ｉ部位であり、単一の下線が引かれた配列は、Ａｓｃ Ｉ部位であり、囲まれたヌクレオチドは、２つの直列の停止コドンの逆方向の相補鎖であり、（Ｎ）_１５は、目的の治療タンパク質をコードする最後の１５のヌクレオチドの相補鎖と同一である。一旦ＰＣＲ産物が増幅されると、この産物はＢｓｕ３６Ｉおよび（Ａｓｃ Ｉ、Ｆｓｅ Ｉ、またはＰｍｅ Ｉ）の１つで切られ、ｐＳｃＮＨＳＡに連結される。

(SEQ ID NO: 1046) where the italicized sequence is the Pme I site and the double underlined sequence is the Fse I site, with a single underline. The sequence shown is the Asc I site, the enclosed nucleotide is the reverse complement of two tandem stop codons, and (N) ₁₅ is the last 15 nucleotides encoding the therapeutic protein of interest. Of the complementary strand. Once the PCR product is amplified, this product is cut with one of Bsu36I and (Asc I, Fse I, or Pme I) and ligated to pScNHSA.

  The presence of the Xho I site in the HSA chimeric leader sequence creates a single amino acid change at the end of the chimeric signal sequence (ie, the HSA-kex2 signal sequence from LDKR (SEQ ID NO: 2139) to LEKR (SEQ ID NO: 2140)). .

(Production of albumin fusion construct containing gene-HSA fusion product)
Similar to the above method, the DNA encoding the therapeutic protein can be PCR amplified using the following primers: HSA leader sequence on the 5 ′ end of the DNA encoding the therapeutic protein, including the Sal I site, of DKR. A 5 ′ primer that adds a polynucleotide encoding the last three amino acids; and a polynucleotide encoding the first few amino acids of mature HSA containing a Cla I site on the 3 ′ end of the DNA encoding the therapeutic protein 3 'primer. For example, the forward primer used to amplify DNA encoding a therapeutic protein is a sequence

（配列番号１０４７）を含み得、ここで下線を引いた配列は、Ｓａｌ Ｉ部位であり、大文字のヌクレオチドは、ＨＳＡリーダー配列の最後の３つのアミノ酸（ＤＫＲ）をコードし、そして（Ｎ）_１５は、目的の治療タンパク質をコードする最初の１５のヌクレオチドと同一である。同様に、治療タンパク質をコードするＤＮＡを逆方向プライマーは、配列

(SEQ ID NO: 1047), where the underlined sequence is the Sal I site, the uppercase nucleotide encodes the last three amino acids (DKR) of the HSA leader sequence, and (N) ₁₅ Is identical to the first 15 nucleotides encoding the therapeutic protein of interest. Similarly, the reverse primer for the DNA encoding the therapeutic protein is the sequence

（配列番号１０４８）であり、ここでイタリック体の配列は、Ｃｌａ Ｉ部位であり、下線が引かれたヌクレオチドは、ＨＳＡ

(SEQ ID NO: 1048), where the italicized sequence is the Cla I site and the underlined nucleotide is HSA

の成熟形態の最初の９アミノ酸をコードするＤＮＡの逆方向相補鎖であり、そして（Ｎ）_１５は、目的の治療タンパク質をコードする最後の１５ヌクレオチドの逆方向の相補鎖である。一旦ＰＣＲ産物が増幅されると、この産物は、Ｓａｌ ＩおよびＣｌａで切られ、Ｘｈｏ ＩおよびＣｌａ Ｉで消化されたｐＳｃＣＨＳＡに連結される。異なるシグナル配列またはリーダー配列が、所望され得、例えば、転化酵素「ＩＮＶ」（Ｓｗｉｓｓ−Ｐｒｏｔ受託Ｐ００７２４）、交配因子α「ＭＡＦ」（Ｇｅｎｂａｎｋ受託番号ＡＡＡ１８４０５）、ＭＰＩＦ（遺伝子配列ＡＡＦ８２９３６）、フィブリン Ｂ（Ｓｗｉｓｓ−Ｐｒｏｔ受託Ｐ２３１４２）、クラステリン（Ｓｗｉｓｓ−Ｐｒｏｔ受託Ｐ１０９０９）、インスリン様増殖因子結合タンパク質４（Ｓｗｉｓｓ−Ｐｒｏｔ受託Ｐ２２６９２）、およびＨＳＡリーダー配列の順列は、当該分野で公知の標準的な方法によって適切なベクターにサブクローニングされ得る。

The reverse complementary strand of DNA encoding the first 9 amino acids of the mature form of (N) ₁₅ and the reverse complement of the last 15 nucleotides encoding the therapeutic protein of interest. Once the PCR product is amplified, it is cut with Sal I and Cla and ligated to pScCHSA digested with Xho I and Cla I. Different signal sequences or leader sequences may be desired, for example, invertase “INV” (Swiss-Prot accession P00724), mating factor α “MAF” (Genbank accession number AAA18405), MPIF (gene sequence AAF82936), fibrin B ( The permutations of Swiss-Prot accession P23142), clusterin (Swiss-Prot accession P10909), insulin-like growth factor binding protein 4 (Swiss-Prot accession P22692), and the HSA leader sequence are appropriate by standard methods known in the art. Can be subcloned into any vector.

(Production of an albumin fusion construct compatible with expression in the yeast S. cerevisiae)
A Not I fragment containing DNA encoding either an N-terminal or C-terminal albumin fusion protein made from pScNHSA or pScCHSA can then be cloned into the Not I site of pSAC35 with the LEU2 selectable marker.
The resulting vector is then yeast S. cerevisiae. used to transform the expression system of C. cerevisiae.

(Example 3: General expression in yeast S. cerevisiae)
Lithium acetate transformation, electroporation, or known in the art, and / or Sambrook, Fritsch, and Maniatis. 1989. “Molecular Cloning: A Laboratory Manual, 2nd edition” 1-3, and Ausubel et al., 2000. According to other methods, such as those described in Massachetts General Hospital and Harvard Medical School “Current Protocols in Molecular Biology”, volumes 1-4, yeast expression compatible expression vectors can be obtained. can be transformed into cerevisiae. This expression vector is transformed into yeast S. cerevisiae by transformation. cerevisiae strains DXY1, D88, or BXP10. Individual transformants can grow for example for 3 days at 30 ° C. in 10 mL YEPD (1% w / v yeast extract, 2% w / v, peptone, 2% w / v, dextrose) Cells can then be collected in the stationary phase after 60 hours of growth. The supernatant can be collected by removing cells at 3000 g for 10 minutes.

  pSAC35 (See Sleep et al., 1990, Biotechnology 8:42 and FIG. 3) contains the complete yeast 2 μm plasmid in addition to the LEU selectable marker and provides replication function, PRB1 promoter, and ADH1 termination signal.

Example 4: General purification of albumin fusion protein expressed from albumin fusion in the yeast S. cerevisiae
In a preferred embodiment, the albumin fusion protein of the invention is a mature form of a therapeutic protein or portion thereof (eg, a mature form of a therapeutic protein listed in Table 1 or a mature mature therapeutic protein shown as SEQ ID NO: Z in Table 2). A mature form of HSA fused to either the N-terminus or the C-terminus of the In one embodiment of the invention, the albumin fusion protein of the invention further comprises a signal sequence that directs the nascent fusion polypeptide in the secretory pathway of the host used for expression. In a preferred embodiment, the signal peptide encoded by the signal sequence is removed and the mature albumin fusion protein is secreted directly into the culture medium. The albumin fusion protein of the present invention is preferably a heterologous signal sequence (eg, a non-natural signal sequence of a particular therapeutic protein) (MAF, INV, Ig, fibrin B, clusterin, insulin-like growth factor binding protein 4, variant HSA leader Sequences (including but not limited to chimeric HSA / MAF leader sequences) or other heterologous signal sequences known in the art. As signal sequences listed in Table 2 and / or signal sequences listed in the section “Fusion protein expression” and / or “Further methods of recombinant and synthetic production of albumin fusion proteins” in the above specification, in particular preferable. In a preferred embodiment, the fusion protein of the invention comprises an N-terminal methionine residue. Polynucleotides (including fragments and / or variants) encoding these polypeptides are also encompassed by the present invention.

  Albumin fusion proteins expressed in yeast as described above can be purified on a small scale on a Dyax peptide affinity column as follows. Supernatants from yeast expressing albumin fusion protein are diafiltered against 3 mM phosphate buffer pH 6.2, 20 mM NaCl and 0.01% Tween 20, to reduce volume and remove dye. The solution is then filtered through a 0.22 μm device. The filtrate is loaded onto a Dyax peptide affinity column. The column is eluted with 100 mM Tris / HCl, pH 8.2 buffer. Peak fractions containing protein are concentrated 5 times, then collected on SDS-PAGE and analyzed.

For large scale purification, the following method may be utilized. Over 2 L of the product is diafiltered and concentrated to 500 mL of 20 mM Tris / HCl pH 8.0. The concentrated protein solution was loaded onto a pre-equilibrated 50 mL DEAE-Sepharose Fast Flow column, the column was washed, and the protein was washed from 0-0.4 M NaCl in 20 mM Tris / HCl, pH 8.0. Elute with a linear gradient of NaCl. These fractions containing protein are pooled and adjusted to pH 6.8 with 0.5 M sodium phosphate (NaH ₂ PO ₄ ). A final concentration of 0.9 M (NH ₄ ) ₂ SO ₄ was added to the protein solution and all solutions were loaded onto a pre-equilibrated 50 mL Butyl650S column. The protein is eluted with a linear gradient of ammonium sulfate (0.9 against 0M (NH ₄ ) ₂ SO ₄ ). These fractions with albumin fusion are pooled again, diafiltered against 10 mM Na ₂ HPO ₄ / citrate buffer pH 5.75 and loaded onto a 50 mL pre-equilibrated SP-Sepharose Fast Flow column. The protein is eluted with a 0-0.5M NaCl linear gradient. Fractions containing the protein of interest are mixed and the buffer is changed to 10 mM Na ₂ HPO ₄ / citric acid pH 6.25 with an Amicon concentrator, whose conductivity is lower than 2.5 mS / cm. The protein solution is loaded onto a 15 mL pre-equilibrated Q-Sepharose high performance column, the column is washed and the protein is eluted with a linear NaCl gradient from 0 to 0.15 M NaCl. The purified protein can then be formulated into a specific buffer composition by buffer exchange.

Example 5: Production of a general construct for mammalian cell transfection
(Production of an albumin fusion construct compatible with expression in mammalian cell lines)
Albumin fusion constructs can be produced in expression vectors for use in mammalian cell culture systems. DNA encoding a therapeutic protein can be cloned N-terminal or C-terminal to HSA in a mammalian expression vector by standard methods known in the art (eg, PCR amplification, restriction digestion, and ligation). Once the expression vector is constructed, transfection into a mammalian expression system can proceed. Suitable vectors are known in the art, including but not limited to pC4 vectors and / or vectors available from Lonza Biologicals, Inc. (Portsmouth, NH).

  DNA encoding human serum albumin is cloned into a pC4 vector creating plasmid pC4: HSA (ATCC Accession No. PTA-3277) suitable for mammalian culture systems. This vector has the gene dihydrofolate reductase “DHFR” allowing selection in the presence of methotrexate.

  The pC4: HSA vector is suitable for the expression of albumin fusion proteins in CHO cells. For expression, in other mammalian cell culture systems, it may be desirable to subclone a fragment containing or consisting of DNA encoding an albumin fusion protein into an alternative expression vector. For example, a fragment comprising or consisting of DNA encoding a mature albumin fusion protein can be subcloned into another expression vector (including but not limited to any mammalian expression vector described herein). obtain.

  In a preferred embodiment, the DNA encoding the albumin fusion construct is obtained from Lonza Biologics, Inc., by means known in the art for expression in NS0 cells. Subcloned into the vector provided by (Portsmouth, NH).

(Generation of albumin fusion constructs containing HSA therapeutic protein fusion products)
pC4: HSA (ATCC accession number PTA-3277) is used to generate an albumin fusion construct where the therapeutic protein moiety is the C-terminus of the mature albumin sequence. For example, DNA encoding a therapeutic protein fragment or variant thereof between the Bsu 36I and Asc I restriction sites of the vector can be cloned. When cloning into Bsu 36I and Asc I, the same primer design used to clone into the yeast vector system (SEQ ID NO: 1045 and SEQ ID NO: 1046) can be utilized (see Example 2).

(Production of albumin fusion construct containing gene HSA fusion product)
Using pC4: HSA (ATCC Accession Number PTA-3277), an albumin fusion construct can be produced in which the therapeutic protein portion is cloned at the N-terminus of the mature albumin sequence. For example, DNA encoding a therapeutic protein having the signal sequence of the therapeutic protein itself between Bam HI (or Hind III) and Cla I of pC4: HSA can be cloned. Kozak sequence when cloned into either Bam HI or Hind III sites

を含み、その後に治療タンパク質をコードするＤＮＡの翻訳開始部分を含むことが好ましい。治療タンパク質が、シグナル配列を含まない場合、治療タンパク質をコードするＤＮＡを、ｐＣ４：ＨＳＡのＸｈｏ ＩとＣｌａ Ｉ部位との間でクローニングし得る。Ｘｈｏ Ｉ部位を用いる場合、以下の５’（配列番号１０５２）および３’（配列番号１０５３）例示的プライマーが、用いられ得る：

Followed by a translation initiation portion of the DNA encoding the therapeutic protein. If the therapeutic protein does not contain a signal sequence, the DNA encoding the therapeutic protein can be cloned between the Xho I and Cla I sites of pC4: HSA. When using the Xho I site, the following 5 ′ (SEQ ID NO: 1052) and 3 ′ (SEQ ID NO: 1053) exemplary primers can be used:

５’プライマー（配列番号１０５２）において、下線が引かれた配列は、Ｘｈｏ部位である；そしてこのＸｈｏ部位およびＸｈｏ Ｉ部位に続くＤＮＡは、天然のヒト血清アルブミンのリーダー配列の最後の７つのアミノ酸をコードする。配列番号１０５２において、「（Ｎ）_１８」は、目的の治療タンパク質をコードする最初の１８ヌクレオチドと同一なＤＮＡである。３’プライマー（配列番号１０５３）において、下線が引かれた配列は、Ｃｌａ Ｉ部位であり、そしてこのＣｌａ Ｉ部位およびそれに続くＤＮＡは、成熟ＨＳＡタンパク質（配列番号１０３８）の最初の１０アミノ酸をコードするＤＮＡの逆方向補完物である。配列番号１０５３において、「（Ｎ）_１８」は、目的の治療タンパク質をコードする最後の１８ヌクレオチドをコードするＤＮＡの逆方向相補鎖である。これらの２つのプライマーを用いて、目的の治療タンパク質をＰＣＲ増幅し得、ＰＣＲ産物を精製し得、Ｘｈｏ ＩおよびＣｌａ Ｉ制限酵素でこの産物を消化し得、そしてｐＣ４：ＨＳＡベクター中のＸｈｏ ＩおよびＣｌａ部位にクローニングし得る。

In the 5 ′ primer (SEQ ID NO: 1052), the underlined sequence is the Xho site; and the DNA following the Xho and Xho I sites is the last seven amino acids of the native human serum albumin leader sequence. Code. In SEQ ID NO: 1052, “(N) ₁₈ ” is DNA identical to the first 18 nucleotides encoding the therapeutic protein of interest. In the 3 ′ primer (SEQ ID NO: 1053), the underlined sequence is a Cla I site, and this Cla I site and subsequent DNA encode the first 10 amino acids of the mature HSA protein (SEQ ID NO: 1038). Is the reverse complement of DNA. In SEQ ID NO: 1053, “(N) ₁₈ ” is the reverse complementary strand of DNA encoding the last 18 nucleotides encoding the therapeutic protein of interest. With these two primers, the therapeutic protein of interest can be PCR amplified, the PCR product can be purified, this product can be digested with Xho I and Cla I restriction enzymes, and Xho I in the pC4: HSA vector. And can be cloned into the Cla site.

If an alternative leader sequence is desired, the native albumin leader sequence can be replaced with a chimeric albumin leader, ie, an HSA-kex2 signal peptide, or an alternative leader by standard methods known in the art. (For example, one skilled in the art will PCR amplify alternative leaders in a predetermined order and maintain the reading frame while subcloning the PCR product into an albumin fusion construct instead of an albumin leader)
(Example 6: General expression in mammalian cell lines)
Albumin fusion constructs produced in expression vectors compatible with expression in mammalian cell lines may be calcium phosphate precipitation, lipofectamine, electroporation, or known in the art and / or Sambrook, Fritsch, and Maniatis 1989 “Molecular Cloning: A Laboratory Manual, 2nd Edition” and Ausubel et al., 2000 Massachusetts General Hospital and Harvard Medical School, “Transformation by Current Protocols in Molecular Biology, et al. Can be transfected into any cell line. The transfected cells are then selected by the presence of a selection agent determined by a selectable marker in the expression vector.

  The pC4 expression vector (ATCC accession number 209646) is a derivative of the plasmid pSV2-DHFR (ATCC accession number 37146). pC4 is a strong promoter, Long Sarcoma Virus Long Terminal Repeats “LTR” (Cullen et al., March 1985, Molecular and Cellular Biology, 438-447) and CytoMegalo Virus et al. : 521-530). This vector also contains the 3 'intron, polyadenylation and termination signal of the rat preproinsulin gene, and the mouse DHFR gene under the control of the SV40 early promoter. Chinese hamster ovary “CHO” cells or other cell lines lacking an active DHFR gene are used for transfection. Transfection of albumin fusion constructs in pC4 into CHO cells by methods known in the art allows expression of the albumin fusion protein in CHO cells, followed by leader sequence cleavage and secretion into the supernatant. The albumin fusion protein is then further purified from the supernatant.

  The pEE12.1 expression vector is available from Lonza Biologicals, Inc. (Portsmouth, NH), which is a derivative of pEE6 (Stephens and Cockett, 1989, Nucl. Acids Res. 17: 7110). This vector contains the complete 5 ′ untranslated region of the Major Immediate Early gene (International Publication No. WO89 / 01036) of the human CytoMegalo Virus “hCMV-MIE” upstream of the promoter, enhancer and sequence of interest, as well as a selective methionine. Glutamine Synthetase gene (Murphy et al., 19991, Biochem J. 227: 277-279; Bebbington et al., 1992, Bio / Technology 10: for the purpose of selection of transfected cells in sulfoximine-containing media: 169-175; U.S. Pat. No. 5,122,464). Transfection of NS0 cells (International Publication No. WO86 / 05807) with albumin fusion constructs made in pEE12.1 by methods known in the art allows expression of albumin fusion proteins in NS0 cells, which leads to leader sequence cleavage. , And secretion into the supernatant follows. The albumin fusion protein is then further purified from the supernatant using the techniques described herein or other methods known in the art.

  The expression of the albumin fusion protein can be analyzed, for example, by SDS-PAGE and Western blot, reverse phase HPLC analysis, or other methods known in the art.

  Stable CHO and NS0 cell lines transfected with an albumin fusion protein are generated by methods known in the art (eg, lipofectamine transfection), eg, as a selectable marker or when glutamine is absent Are selected with 100 nM methotrexate for vectors with dihydrofolate reductase through growth of Serum-containing antibodies whose expression levels are directed, for example, firstly using anti-HSA serum as the first antibody or secondly as the first antibody to the therapeutic protein portion of a given albumin fusion protein And can be tested by immunoblotting.

  Expression levels are tested by immunoblot detection with anti-HSA serum as the first antibody. Specific production rate is determined via ELISA, where the capture antibody can be a monoclonal antibody directed to the therapeutic protein portion of the albumin fusion and the detection antibody can be a monoclonal anti-HSA biotinylated antibody (or vice versa). This is followed by horseradish peroxidase / streptavidin binding and analysis according to the manufacturer's protocol.

Example 7 General Purification of Albumin Fusion Protein Expressed from Albumin Fusion Protein in Mammalian Cell Lines
In a preferred embodiment, the albumin fusion protein of the invention is a therapeutic protein or portion thereof (eg, a mature form of a therapeutic protein listed in Table 1 or a mature form of a therapeutic protein shown as SEQ ID NO: Z in Table 2). Includes mature forms of HSA fused to either the N-terminus or C-terminus of the mature form. In one embodiment of the invention, the albumin fusion protein of the invention further comprises a signal sequence that directs the nascent fusion polypeptide in the host secretory pathway used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed and the mature albumin fusion protein is secreted directly into the culture medium. The albumin fusion protein of the present invention is preferably a heterologous signal sequence (eg, a non-natural signal sequence of a particular therapeutic protein) (MAF, INV, Ig, fibrin B, clusterin, insulin-like growth factor binding protein 4, variant HSA leader Sequences (including but not limited to chimeric HSA / MAF leader sequences) or other heterologous signal sequences known in the art. As signal sequences listed in Table 2 and / or signal sequences listed in the section “Fusion protein expression” and / or “Further methods of recombinant and synthetic production of albumin fusion proteins” in the above specification, in particular preferable. In a preferred embodiment, the fusion protein of the invention further comprises an N-terminal methionine residue. Polynucleotides (including fragments and / or variants) encoding these polypeptides are also encompassed by the present invention.

  Albumin fusion proteins from mammalian cell line supernatants are purified according to different protocols according to the expression system used.

(Purification from CHO and 293 cell lines)
Purification of albumin fusion protein from CHO cell supernatant or from transiently transfected 293T cell supernatant involves initial capture with anionic HQ resin using sodium phosphate buffer and phosphate gradient elution. This is followed by affinity chromatography on a Blue Sepharose FF column using salt gradient elution. Blue Sepharose FF removes major BSA / fetuin contaminants. Further purification of the Poros PI 50 resin using a phosphate gradient can remove and reduce endotoxin contaminants and concentrate albumin fusion proteins.

(Purified from NS0 cell line)
Purification of albumin fusion protein from NS0 cell supernatant may include Q-Sepharose anion exchange chromatography followed by SP-Sepharose purification using step elution followed by Phenyl-650M using step elution, and finally This is followed by diafiltration.

  The purified protein can then be formulated by buffer exchange.

(Example 8: Construct ID 1966, EPO-HSA, production)
Construct ID 1966 (pC4.EPO: M1-D192.HSA) encodes an EPO-HSA fusion protein, which is the amino terminus of the mature form of HSA cloned into the EPO native leader sequence and the mammalian expression vector pC4. Contains the mature EPO protein except the final Arg residue (ie, M1-D192) fused to

(Cloning of EPO cDNA for construct 1966)
DNA encoding EPO was amplified using primers EPO1 and EPO2 described below, cut with Bam HI / Cla I, and ligated into Bam HI / Cla I cut pC4: HSA. Construct ID number 1966 encodes an albumin fusion protein comprising a mature HSA protein followed by a mature HSA protein (see SEQ ID NO: 297 for construct 1966 in Table 2).

  Two oligonucleotides (EPO1 and EPO2) suitable for PCR amplification of polynucleotides encoding full-length EPO containing the natural leader sequence were synthesized.

ＥＰＯ１は、Ｂａｍ ＨＩクローニング部位（斜体で示される）を組み込み、全長ＥＰＯのＯＲＦの最初の３５アミノ酸をコードするＤＮＡの前にｋｏｚａｋ配列（二重下線を引いて示される）を結び付ける。ＥＰＯ２において、下線を引かれた配列は、Ｃｌａ Ｉ部位であり；そしてＣｌａ Ｉ部位およびそれに続くＤＮＡは、成熟ＨＳＡタンパク質の最初の１０個のアミノ酸をコードするＤＮＡの逆相補体である（配列番号１０３８）。ＥＰＯ２において太字の核酸配列は、ＥＰＯの全長形態のアミノ酸残基Ｇｌｕ−１８６〜Ａｓｐ１９２をコードする最後の２２個のヌクレオチドの逆相補体である。これらの２つのプライマーを使用して、最終Ａｒｇ残基を除いた全長ＥＰＯタンパク質を、ＰＣＲ増幅した。アニーリング温度および伸長温度ならびにアニーリング回数および伸長回数が、各々の特異的なプライマー対およびテンプレートについて経験的に決定されなければならない。
ＰＣＲ産物を精製し（例えば、Ｗｉｚａｒｄ ＰＣＲ Ｐｒｅｐｓ ＤＮＡ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｓｙｓｔｅｍ（Ｐｒｏｍｅｇａ Ｃｏｒｐ））、続いてＢａｍ ＨＩおよびＣｌａ Ｉで消化した。そしてゲル電気泳動によるＢａｍ ＨＩ−Ｃｌａ Ｉフラグメントのさらなる精製の後、この産物をＢａｍ ＨＩ／Ｃｌａ Ｉで消化されたｐＣ４：ＨＳＡにクローニングし、構築物番号１９６６を産生した。
さらに、発現されたアルブミン融合タンパク質のＮ末端の、アミノ酸配列決定による分析により、予測されたＥＰＯ配列の存在を確認した（以下を参照のこと）。

EPO1 incorporates a Bam HI cloning site (shown in italics) and binds the kozak sequence (shown with double underlining) in front of the DNA encoding the first 35 amino acids of the full-length EPO ORF. In EPO2, the underlined sequence is the Cla I site; and the Cla I site and the DNA that follows is the reverse complement of the DNA encoding the first 10 amino acids of the mature HSA protein (SEQ ID NO: 1038). The bold nucleic acid sequence in EPO2 is the reverse complement of the last 22 nucleotides encoding amino acid residues Glu-186 to Asp192 in the full-length form of EPO. Using these two primers, the full length EPO protein excluding the final Arg residue was PCR amplified. The annealing temperature and extension temperature as well as the number of annealing times and extension times must be determined empirically for each specific primer pair and template.
The PCR product was purified (eg, Wizard PCR Preps DNA Purification System (Promega Corp)) and subsequently digested with Bam HI and Cla I. Then, after further purification of the Bam HI-Cla I fragment by gel electrophoresis, this product was cloned into pC4: HSA digested with Bam HI / Cla I to produce construct number 1966.
In addition, the presence of the predicted EPO sequence was confirmed by analysis by amino acid sequencing of the N-terminus of the expressed albumin fusion protein (see below).

The EPO albumin fusion protein of the invention is preferably an HSA fused to either the N-terminus or C-terminus of the mature form of EPO lacking the final Arg residue (ie, Ala-28 to Asp-192). Includes the mature form (ie Asp-25-Leu-609). In one embodiment of the invention, the EPO albumin fusion protein of the invention further comprises a signal sequence that directs the nascent fusion polypeptide in the secretory pathway of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed and the mature EPO albumin fusion protein is secreted directly into the culture medium. The EPO albumin fusion protein of the present invention includes heterologous signal sequences (MAF, INV, Ig, Fibulin B, Clusterin, insulin-like growth factor binding protein 4, modified HSA leader sequences (including chimeric HSA / MAF leader sequences, Or other heterologous signal sequences known in the art, including but not limited to). In a preferred embodiment, the EPO albumin fusion protein of the invention comprises a native EPO signal sequence. In a further preferred embodiment, the EPO albumin fusion protein of the present invention further comprises an N-terminal methionine residue. Polynucleotides (including fragments and / or variants) encoding these polypeptides are also encompassed by the present invention.
(Expression and purification of construct ID 1966)
(Expression in either 293T cells or CHO cells)
Construct 1966 was transfected into 293T cells by methods known in the art (eg, lipofectamine transfection) and selected with 100 nM methotrexate (see Example 6). Expression levels were tested by immunoblot detection using anti-HSA serum as the primary antibody and specific production rates were determined by monoclonal anti-human EPO antibody for capture (Research Diagnostics, Inc.) and Biotrend monoclonal for detection. Determined via ELISA using anti-HSA biotinylated antibody followed by horseradish peroxidase / streptavidin binding and analysis.

(Purification from 293T cell supernatant)
The 293T cell supernatant containing secreted EPO-HSA fusion protein expressed in construct ID numbers 1966 to 293T cells was purified as described in Example 7. Specifically, initial capture is performed with anionic HQ-50 resin at pH 7.2 using step elution, followed by blue sepharose reusing step elution at pH 7.2. Performed using column chromatography. Pooled fractions were passed through HQ-50 resin using step elution. The eluted sample was loaded onto a Phenyl-650M column and eluted with a gradient elution at pH 7.2. The eluted sample was passed through HQ-50 resin three times. The fraction of interest was diafiltered into 50 mM Na ₂ HPO ₄ +200 mM NaCl (pH 7.2). N-terminal sequencing yielded the amino terminal sequence of the mature form of EPO (ie APPRLI). A protein with an approximate molecular weight of 90 kDa was obtained. A final yield of 0.42 mg protein per liter 293T cell supernatant was obtained.

(Purification from CHO cell supernatant)
Cell supernatant containing EPO albumin fusion protein expressed from construct number 1966 in CHO cells was purified as described in Example 7. Specifically, initial capture of a concentrated 1.4 L sample was performed using an anionic Poros using sodium phosphate buffer and phosphate gradient elution (0-100 mM sodium phosphate, pH 7.2). Performed with HQ 50 resin. Prior to loading on the column, the sample was diluted to 3 mM phosphate as in the further column chromatography purification until the conductivity was less than 5.0 mS. HQ resin was equilibrated with 10 mM sodium phosphate (pH 7.2 before sample loading). EPO-HSA was eluted with 20 mS or 50 mS sodium phosphate.
The second purification step included affinity chromatography. The combined fractions from the previous HQ resin, adapted for conductivity below 5 mS using 3 mM phosphate buffer (pH 7.2), were equilibrated with 125 mM NaCl, 15 mM sodium phosphate (pH 7.2) Blue It was mounted on a Sepharose FF column. A salt gradient of 0-3M NaCl eluted EPO-HSA between 0.5M and 1.0M NaCl. Blue Sepharose FF removes major BSA / fetuin contaminants. In a third column containing Poros PI50 resin that removes and reduces endotoxin contaminants and concentrates the EPO-HSA protein, the conductivity of the desired fractions is again adapted and the pooled fractions are It was loaded. The resin was equilibrated with 25 mM NaCl, 10 mM sodium phosphate (pH 7.2). EPO-HSA was eluted with a 10 mM-100 mM phosphate gradient. The final buffer composition was 100 mM NaCl, 20 mM Na ₂ HPO ₄ (pH 7.2). An approximate protein molecular weight of 87.7 kDa was obtained. A final yield of 8.9 mg protein per liter supernatant was obtained. N-terminal sequencing yielded the sequence APPRL corresponding to the amino terminus of the mature form of EPO.

(In vitro TF-1 cell proliferation assay)
(Method)
The biological activity of the EPO albumin fusion protein can be measured in an in vitro TF-1 cell proliferation assay. The TF1 cell line was established by Kitamura et al. (Kitamura, T. et al., 1989, J. Cell. Physiol., 140: 323-334). TF-1 cells were derived from a heparinized bone marrow aspirate sample from a 35 year old Japanese male with severe pancytopenia. The TF-1 cell line provides a good system for investigating the proliferation and differentiation of bone marrow progenitor cells as a result of responsiveness to multiple cytokines.

TF-1 cell proliferation assay (Kitamura, T. et al., 1989, J. Cell. Physiol., 140: 323-334): human TF-1 cells (ATCC number CRL-2003) were analyzed with 10% FBS, 1X pen- Spread strep, 1 × L-glutamine, and 2 ng / mL human GM-CSF in RPMI 1640 medium to 1 × 10 ⁶ cells / mL. Cells were passaged every 2-3 days by diluting 1:10 or 1:20 in fresh medium. On the day of assay start, the cells are washed 3 times with a 50 mL volume of RPMI 1640/10% FBS, the GM-CSF is removed and resuspended at 1 × 10 ⁵ cells / mL in RPMI 1640/10%. Cells are plated at 10,000 cells / well in TC-treated 96 well plates with flat bottoms. Three-fold serial dilutions of the control protein are made in RPMI in the range of 10 U / mL to 0.001 U / mL (final concentration), where 1 U = 10 ng protein; 0.1 mL of each dilution is added to each Add to triplicate wells containing cells for a final volume of 0.2 mL in the wells. Cell proliferation and albumin fusion proteins in response to control proteins are determined by measuring ³ H-thymidine (0.5 uCi / well) uptake. This assay is performed at an incubation time of 24 hours, 48 hours, or 72 hours prior to the addition of ³ H-thymidine, and for 4-24 hours after the addition. Only the molecular weight portion of the albumin fusion protein is effectively a therapeutic protein molecule (ie, the therapeutic protein portion of the fusion) if the diluent can also be adapted to a molar ratio.

In vitro TF-1 cell proliferation assay for albumin fusion protein encoded by construct 1966
(Method)
TF-1 cell proliferation assay: Human TF-1 cells (ATCC No. CRL-2003) were treated with 10% FBS, 1X pen-strep, 1X L-glutamine, and 2 ng / mL human GM-CSF in RPMI medium at 1 × 10. Spread to a maximum density of ⁶ cells / mL. Cells were passaged every 2-3 days by diluting 1:10 or 1:20 in fresh medium. On the day of assay start, cells were washed 3 times with a 50 mL volume of RPMI 1640/10% FBS to remove GM-CSF and resuspended at 1 × 10 ⁵ cells / mL in RPMI 1640/10%. Cells were plated at 10,000 cells / well in TC-treated 96-well plates with flat bottoms. Three-fold serial dilutions of hrEPO (R & D Systems; Research Diagnostics Inc., RDI) in the RPMI 1640/10% range from 10 U / mL to 0.001 U / mL (final concentration), where 1 U = 10 ng protein. 0.1 mL of each dilution was added to triplicate wells containing cells for a final volume of 0.2 mL in each well. Cell proliferation response and EPO albumin fusion protein in response to hrEPO were determined by measuring ³ H-thymidine (0.5 μCi / well) uptake. This assay was performed at an incubation time of 24, 48, or 72 hours prior to the addition of ³ H-thymidine and for 4-24 hours after the addition. Only 1/3 of the molecular weight of the EPO albumin fusion protein is effectively an EPO molecule if the diluent can also be adapted to the molar ratio.

(result)
EPO-HSA albumin derived proteins derived from supernatants from 293T cells expressing construct 1966 or CHO cells expressing construct 1966 were tested in the above assay for EPO activity. On average, an EC50 greater than 5 times rhEPO was established (see FIG. 4).

(In vivo mouse Harlan for measuring hematocrit)
(Method)
This mouse model provides a means to measure in vivo protein therapeutic activity by measuring effects on the hematocrit.

  In vivo mouse models (ie, 6-8 week old female DBA / 2NHsd mice (Harlan)) were intravenously, intraperitoneally, or subcutaneously, daily for 7 days, or at 2 μg / kg every other day. It was established to monitor the effect on hematocrit in the administration of albumin fusion protein at control protein or 30 μg / kg, and at other concentrations, and at other concentrations. Hematocrit is determined by piercing the tail vein with a needle, collecting blood in a heparinized microcapillary tube, and then spinning the tube over the duration of the experiment. Also, for a specific experiment, the spleen is collected and weighed. Other dosing schedules are known within the art and can be readily adapted for use in this assay.

(The activity of the albumin fusion protein encoded by construct 1966 can be assayed using an in vivo Harlan mouse model to measure hematocrit.)
Using an in vivo mouse model of 6-8 week old female DBA / 2NHsd mice (Harlan), 0.5 μg / kg, 1.5 μg / kg, 4 days on days 0, 2, 4 and 6 Administration of rhEPO (Research Diagnostics, Inc., Catalok No. RDI-PB11965) at dosages of 5 μg / kg, 12 μg / kg, and subcutaneous, “SC” on days 0, 2, 4 and On day 6, the extent of EPO activity was monitored in the administration of purified albumin fusion protein encoded by construct 1966 at concentrations of 2 μg / kg, 6 μg / kg, 18 μg / kg, and 54 μg / kg.
Hematocrit was determined by piercing the tail vein with a needle on days 0 and 7, collecting blood in heparinized microcaileries tubes, and subsequently spinning the tubes over the duration of the experiment. . The higher dosage of EPO albumin fusion protein is approximately equimolar compared to the control recombinant human EPO, “rhEPO” (Research Diagnostics, Inc., Catalok number RDI-PB11965).

(result)
There was a significant increase in the 0-7 day hematocrit treated with either recombinant human EPO or EPO albumin fusion protein (see Example 5). However, the EPO albumin fusion protein encoded by construct 1966 appeared to have a more intense effect at the hematocrit level than the rhEPO control.
Subcutaneous administration of 3 doses / week of 52 μg / kg or 1 dose / week of 156 μg / kg of EPO albumin fusion protein encoded by construct 1966 is greater than or equal to 40% change in hematocrit on days 0-8 Caused a change equal to (see FIG. 6). The% change in hematocrit is kept close to 40% for the triple dose, or from near 30% to less than 10% for a 3 dose subcutaneous dose of 12 μg / kg of rhEPO during one week. Contrary to the decrease, it is either suppressed to 20% or less for a single dose on day 14. Elevated hematocrit was kept with the EPO albumin fusion protein encoded by construct 1966 over a period of one week after the last subcutaneous administration compared to hematocrit levels induced by rhEPO protein that fall to more normal levels Seems to be.

  DBA mice injected intravenously with 150 μg / kg of albumin fusion protein removed this EPO albumin fusion protein 7 times more slowly than rhEPO.

(Example 9: Construct ID 1981, HSA-EPO, production)
Construct ID 1981 (pC4.EPO: HSA-EPO.A28-D192) encodes an EPO albumin fusion protein that contains the native HSA leader sequence and the amino form of the mature form of EPO cloned into the mammalian expression vector pC4. Includes DNA excluding the final Arg residue fused to the ends.

(Cloning of EPO cDNA for construct 1981)
DNA encoding EPO was amplified using the primers EPO3 and EPO4 described below, cut with Bsu 36I / Asc I, and ligated into Bsu 36I / Asc I cut pC4: HSA. Construct ID number 1981 encodes an albumin fusion protein including the mature form of HSA and the mature form of EPO, Ala 28-Asp192 (Genbank Access AAA52400).

  Two oligonucleotides (EPO3 and EPO4) appropriate for PCR amplification of the polynucleotide encoding the mature form of EPO (see SEQ ID NO: X for construct 1981 in Table 2) were synthesized.

ＥＰＯ３は、Ｂｓｕ ３６Ｉクローニング部位（下線で示される）およびＥＰＯの成熟形態の最初の８アミノ酸をコードするＨＳＡの最後の４アミノ酸残基および２６ヌクレオチド（イタリック体）をコードするヌクレオチドを組み込む。ＥＰＯ４において、Ａｓｃ Ｉ部位は、下線を引かれ（配列番号８０６）、そして最後の２８ヌクレオチド（イタリック体）は、最終Ａｒｇ残基を除いたＥＰＯの最後の９個のアミノ酸残基をコードするＤＮＡの逆相補体である（一般的な構築物クローニングについては実施例５を参照のこと）。これらのプライマーを使用して精製されたＰＣＲ増幅体を精製し、Ｂｓｕ ３６Ｉ／Ａｓｃ Ｉ制限酵素で消化し、そしてｐＣ４：ＨＳＡベクターのＢｓｕ ３６Ｉ／Ａｓｃ Ｉ部位へクローニングした。
ＰＣＲ産物を精製し（例えば、Ｗｉｚａｒｄ ＰＣＲ Ｐｒｅｐｓ ＤＮＡ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｓｙｓｔｅｍ（Ｐｒｏｍｅｇａ Ｃｏｒｐ））、続いてＢｓｕ ３６ＩおよびＡｓｃ Ｉで消化した。そしてゲル電気泳動によるＢｓｕ ３６Ｉ−Ａｓｃ Ｉフラグメントのさらなる精製の後、この産物をＢｓｕ ３６Ｉ／Ａｓｃ Ｉで消化されたｐＣ４：ＨＳＡにクローニングし、構築物番号１９８１を産生した。
さらに、発現されたアルブミン融合タンパク質のＮ末端の、アミノ酸配列決定による分析は、予測されたＨＳＡ配列の存在を確認した（以下を参照のこと）。

EPO3 incorporates a Bsu 36I cloning site (shown underlined) and the last 4 amino acid residues of HSA encoding the first 8 amino acids of the mature form of EPO and nucleotides encoding 26 nucleotides (italicized). In EPO4, the Asc I site is underlined (SEQ ID NO: 806) and the last 28 nucleotides (italicized) are the DNA encoding the last 9 amino acid residues of EPO, excluding the final Arg residue. (See Example 5 for general construct cloning). The PCR amplifications purified using these primers were purified, digested with Bsu 36I / Asc I restriction enzyme and cloned into the Bsu 36I / Asc I site of the pC4: HSA vector.
The PCR product was purified (eg, Wizard PCR Preps DNA Purification System (Promega Corp)) followed by digestion with Bsu 36I and Asc I. And after further purification of the Bsu 36I-Asc I fragment by gel electrophoresis, this product was cloned into pC4: HSA digested with Bsu 36I / Asc I to produce construct number 1981.
Furthermore, analysis by amino acid sequencing of the N-terminus of the expressed albumin fusion protein confirmed the presence of the predicted HSA sequence (see below).

The EPO albumin fusion protein of the invention is preferably an HSA fused to either the N-terminus or C-terminus of the mature form of EPO lacking the final Arg residue (ie, Ala-28 to Asp-192). Includes the mature form (ie Asp-25-Leu-609). In one embodiment of the invention, the EPO albumin fusion protein of the invention further comprises a signal sequence that directs the nascent fusion polypeptide in the secretory pathway of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed and the mature EPO albumin fusion protein is secreted directly into the culture medium. The EPO albumin fusion protein of the present invention includes heterologous signal sequences (MAF, INV, Ig, Fibulin B, Clusterin, insulin-like growth factor binding protein 4, modified HSA leader sequences (including chimeric HSA / MAF leader sequences, Or other heterologous signal sequences known in the art, including but not limited to). In a preferred embodiment, the EPO albumin fusion protein of the invention comprises a native EPO signal sequence. In a further preferred embodiment, the EPO albumin fusion protein of the present invention further comprises an N-terminal methionine residue. Polynucleotides (including fragments and / or variants) encoding these polypeptides are also encompassed by the present invention.
(Expression and purification of construct ID 1981)
(Expression of CHO cells)
Construct 1981 was transfected into CHO cells as described in Examples 6 and 8. Expression levels and specific production rates were determined as described in Example 8.

(Purification from CHO supernatant)
Cell supernatants containing EPO albumin fusion protein expressed in CHO cells from construct ID number 1981 were purified as described in Examples 7 and 8. N-terminal sequencing yielded DAHKS (amino terminal sequence of the mature form of HSA). 14 mg of protein was obtained per liter of supernatant. An approximate molecular weight of 85.7 kDa was obtained.

(In vitro TF-1 cell proliferation assay for construct 1981)
(Method)
The in vitro TF-1 cell proliferation assay for the EPO albumin fusion protein encoded by construct 1981 was previously described in the subsection beginning in Example 8 “In vitro TF-1 cell proliferation assay for construct 1966”. Executed.

(result)
Supernatants from CHO cells expressing construct 1981 were purified over 90% for HSA-EPO albumin fusion protein and tested in the assay as described in Example 8. On average, an EC50 greater than 5 times that of rhEPO was established (see Examples 4 and 7).
(The activity of construct 1981 can be assayed using the in vivo mouse model Harlan to measure hematocrit)
(Method)
The in vivo Harlan mouse model was used to measure hematocrit levels in subcutaneous administration of either control rhEPO or EPO albumin fusion protein encoded by construct 1981. This assay was performed as previously described in Example 8 under the subsection “Activity of construct 1966 can be assayed using the in vivo mouse model Harlan to measure hematocrit”.

(result)
There was a significant increase in hematocrit from day 0-7 for animals treated with either rhEPO or EPO albumin fusion protein (see FIG. 5). However, the EPO albumin fusion protein encoded by construct 1981 appeared to have a more intense effect at the hematocrit level than the rhEPO control.

  DBA mice injected intravenously at 150 μg / kg of EPO albumin fusion protein encoded by albumin fusion construct 1981 removed this EPO albumin fusion protein 7 times more slowly than rhEPO.

(Example 10: Construct ID 1997, HSA-EPO, production)
Construct ID 1997 (pEE12.1: EPO M1-D192.HSA) contains DNA encoding the EPO albumin fusion protein, which fusion protein contains the full length EPO protein (including the native leader sequence) excluding the final Arg residue, It has M1-D192 and is fused to the amino terminus of the mature form of HSA cloned into the mammalian expression vector pEE12.1.

(Cloning of EPO cDNA for Construct 1997)
DNA encoding EPO was amplified using primers EPO5 and EPO6, described below, cut with Eco RI / Cla I, and Eco RI / Cla I cut pcDNA3 (Invitrogen Corporation, 1600 Faraday Ave, Carlsbad, CA 92008). pcDNA3. EPO M1-D192. HSA was digested with Eco RI / Hind III to give EPO M1-D192. Release the HSA expression cassette fragment and digest with Eco RI / Hind III digested pEE12.1. Cloned into. Construct ID number 1997 encodes the albumin fusion protein including the leader sequence and the mature form of EPO, followed by the mature EPO protein (see SEQ ID NO: Y in Table 2 for construct 1997).

  Two oligonucleotides (EPO5 and EPO6) suitable for PCR amplification of polynucleotides encoding EPO (SEQ ID NO: X, Table 2 for construct 1997), EPO5 and EPO6 were synthesized.

ＥＰＯ５は、Ｅｃｏ ＲＩ部位（イタリック体で示される）およびｋｏｚａｋ配列（下線で示される）を組み込み、続いて、全長ＥＰＯのＯＲＦの最初の３５アミノ酸をコードするＤＮＡを組み込む。ＥＰＯ６において、イタリック体の配列は、Ｃｌａ Ｉ部位であり、下線を引かれた配列は、ＨＳＡタンパク質の成熟形態の最初の最初の９アミノ酸をコードするＤＮＡの逆相補体であり（ＤＡＨＫＳＥＶＡＨ、配列番号１１０６）、そしてＨＳＡの逆相補体に続く配列は、最後のＡｒｇ−１９３アミノ酸を含まないＥＰＯの最後の７アミノ酸をコードする、最後の２３ヌクレオチドの逆相補体である。これらの２つのプライマーを使用して、全長ＥＰＯタンパク質をコードするＤＮＡを、実施例８のようにＰＣＲ増幅した。

EPO5 incorporates an Eco RI site (shown in italics) and a kozak sequence (shown in underline), followed by DNA encoding the first 35 amino acids of the full-length EPO ORF. In EPO6, the italicized sequence is the Cla I site and the underlined sequence is the reverse complement of DNA encoding the first 9 amino acids of the mature form of the HSA protein (DAHKSEVAH, SEQ ID NO: 1106), and the sequence following the reverse complement of HSA is the last 23 nucleotide reverse complement encoding the last 7 amino acids of EPO without the last Arg-193 amino acid. Using these two primers, DNA encoding the full-length EPO protein was PCR amplified as in Example 8.

The PCR product was purified and subsequently digested with Eco RI and Cla I. This product was then cloned into Eco RI / Cla I digested pcDNA3 after further purification of the Eco RI-Cla I fragment by gel electrophoresis. An Eco RI / Hind III fragment containing the expression cassette was transformed into pcDNA3. EPO. M1-D192. Generated from HSA and subcloned into EcoRI / HindIII digested pEE12.1, resulting in construct number 1997.
Furthermore, analysis of the N-terminus of albumin fusion protein by amino acid sequencing confirmed the presence of the predicted EPO sequence. (See below).

The EPO albumin fusion protein of the invention is preferably an HSA fused to either the N-terminus or C-terminus of the mature form of EPO lacking the final Arg residue (ie, Ala-28 to Asp-192). Includes the mature form (ie Asp-25-Leu-609). In one embodiment of the invention, the EPO albumin fusion protein of the invention further comprises a signal sequence that directs the nascent fusion polypeptide in the secretory pathway of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed and the mature EPO albumin fusion protein is secreted directly into the culture medium. The EPO albumin fusion protein of the present invention includes heterologous signal sequences (MAF, INV, Ig, Fibulin B, Clusterin, insulin-like growth factor binding protein 4, modified HSA leader sequences (including chimeric HSA / MAF leader sequences, Or other heterologous signal sequences known in the art, including but not limited to). In a preferred embodiment, the EPO albumin fusion protein of the invention comprises a native EPO signal sequence. In a further preferred embodiment, the EPO albumin fusion protein of the present invention further comprises an N-terminal methionine residue. Polynucleotides (including fragments and / or variants) encoding these polypeptides are also encompassed by the present invention.
(Expression and purification of construct ID 1997)
(Expression of NS0 cells)
Construct 1997 was transfected into NS0 cells as described in Example 6. Expression levels and specific production rates were determined as described in Example 8.
(Purification from NS0 cell supernatant)
Purification of albumin fusion protein from 500 mL cell supernatant from NS0 cells transfected with construct 1997 was performed using Q-Sepharose anion exchange chromatography at pH 7.4 using a 0-1 M NaCl gradient in 20 mM Tris-HCl. Followed by Poros PI 50 anion exchange chromatography at pH 6.5 using a 5-40 mM sodium citrate gradient, and final buffer concentrations of 10 mM citric acid (pH 6.5) and 140 mM NaCl (see Example 7). Diafiltration for 6DV to (see)). N-terminal sequencing should yield the sequence APPRLI, which is the amino terminus of the mature form of EPO. This protein has an approximate MW of 87.7 kDa. A final yield of 52.2 mg protein per liter of supernatant was obtained.

For larger scale purification (eg, 50 L NS0 cell supernatant), it can be concentrated to about 8-10 L. The concentrated sample was then passed through a Q-Sepharose anion exchange column (10 × 19 cm, 1.5 L) using a step elution consisting of 50 mM NaOAc (pH 6.0) and 150 mM NaCl at pH 7.5. Can be done. The eluted sample can then be virus infected with 0.75% Triton-X 100 for 60 minutes at room temperature. SDR-reverse phase chromatography (10 cm × 10 cm, 0.8 L) is then used at pH 6.0 with 50 mM NaOAc and 150 mM NaCl, or vice versa, and the sample is loaded onto an SP-Sepharose column at pH 4.8. At 50 mM NaOAc (pH 6.0) and 150 mM NaCl. Subsequently, DV50 is filtered to remove all virus components. Subsequently, Phenyl-650M chromatography (20 cm × 12 cm, 3.8 L) was used with step elution consisting of 350 mM (NH ₄ ) ₂ SO ₄ and 50 mM NaOAc, or step elution consisting of 50 mM NaOAc (pH 6.0). At pH 6.0. Diafiltration for 6-8 DV was performed using a buffer of the desired final concentration of buffer (10 mM Na ₂ HPO ₄ +58 mM sucrose + 120 mM NaCl (pH 7.2) or 10 mM citric acid (pH 6.5) and 140 mM NaCl. Allows exchange to either.
(In vitro TF-1 cell proliferation assay for construct 1997)
(Method)
The in vitro TF-1 cell proliferation assay for the EPO-HSA albumin fusion protein encoded by construct 1997 is as described in the subsection beginning in Example 8 “In vitro TF-1 cell proliferation assay for construct 1966”. Run before.

Supernatants from NS0 cells expressing construct 1997 were purified over 90% for EPO-HSA albumin fusion protein and tested in the assay as described in Example 8. On average, an EC50 greater than 5 times rhEPO was established (see Example 7).
(The activity of construct 1997 can be assayed using the in vivo mouse model Harlan to measure hematocrit)
(Method)
In vivo Harlan mouse model measures hematocrit levels in subcutaneous administration of either control rhEPO or EPO albumin fusion protein encoded by construct 1981 at various dosages at 0, 2, 4, and 6 days Used for. This assay was performed as previously described in Example 8 under the subsection “Activity of construct 1966 can be assayed using the in vivo mouse model Harlan to measure hematocrit”. Hematocrit was determined on day 0, day 8 and day 14.

(result)
There was a significant increase in hematocrit from day 0 to day 8 for animals treated with either rhEPO or EPO albumin fusion protein (see FIG. 8). However, as in the case of the EPO albumin fusion protein encoded by construct 1966, 3 doses / week of subcutaneous administration of 52 μg / kg of EPO albumin fusion protein encoded by construct 1997 resulted in 12 μg / kg subcutaneous of rhEPO per week. Contrary to the decline of 30% to less than 10% for the three doses of the dose level, it caused a near 30% change in the hematocrit from day 8 to day 8 which was reduced to less than 15% on day 14.

  DBA mice injected intravenously with 150 μg / kg of the EPO albumin fusion protein encoded by the EBO albumin fusion construct removed the EPO albumin fusion protein 7 times more slowly than rhEPO.

(Example 11: Construct ID 2294, EPO-HSA, production)
Construct ID 2294 (pC4.EPO R140G.HSA) encodes an EPO albumin fusion protein, which has the full-length EPO protein (including native leader sequence), ie M1-D192, and the mammalian expression vector pC4 The DNA excluding the final Arg residue fused to the amino terminus of the mature form of HSA cloned in

(Cloning of EPO cDNA for construct 2294)
Construct ID number 2294 encodes an albumin fusion protein comprising a leader sequence and a mature form of EPO followed by a mature HSA protein. Construct ID number 2294 was generated by using construct ID number 1966 (ie, pC4: EPO.M1-D192.HSA) as a template in a two-step PCR method.

  Four oligonucleotides (EPO7, EPO8, EPO9 and EPO10) suitable for PCR amplification of polynucleotides encoding EPO (SEQ ID NO: X, for construct 2294, Table 2) were synthesized.

ＰＣＲ増幅の最初の段階で、構築物１９６６の最初のＮ末端フラグメントおよびＣ末端フラグメントが、独立して増幅された。Ｎ末端フラグメントを、プライマーＥＰＯ７およびＥＰＯ８を用いて生成した。ＥＰＯ７は、Ｂａｍ ＨＩ（イタリック体で示される）を組み込み、そしてｋｏｚａｋ配列（下線で示される）、続いて全長ＥＰＯのＯＲＦの最初の６アミノ酸をコードする最初の１８ヌクレオチドを有する。ＥＰＯ８プライマーは、アミノ酸１４０でのＡｒｇ残基をコードするコドンＣＧＡ（太字で強調される）が、Ｇｌｙ残基をコードするコドンＧＧＡに変更される。Ｃ末端フラグメントを、プライマーＥＰＯ９およびＥＰＯ１０を用いて生成した。ＥＰＯ９において、下線が引かれた配列は、Ｃｌａ Ｉ部位であり、そしてＣｌａ Ｉ部位およびそれに続くＤＮＡは、成熟ＨＳＡタンパク質の最初の１０アミノ酸をコードするＤＮＡの逆相補体である。ＥＰＯ９において、最後の５ヌクレオチドは、全長ＥＰＯにおける最後の５ヌクレオチドの逆相補体に対応し、これは最終Ａｒｇ−１９３残基を欠失する。ＥＰＯ１０プライマーは、アミノ酸１４０でのＡｒｇ残基をコードするコドンＣＧＡ（太字で強調される）が、Ｇｌｙ残基をコードするコドンＧＧＡに変更されることを除いて、ＥＰＯの全長形態の１３６〜１４３アミノ酸をコードする核酸配列を含む。ＰＣＲ増幅の第２の段階において、プライマーＥＰＯ７およびＥＰＯ９が、Ａｒｇ−１４０を有する全長ＥＰＯを、反応混合物が、ＰＣＲ増幅されたＮ末端フラグメントおよびＰＣＲ増幅されたＣ末端フラグメントの両方を含んだ、Ｇｌｙ変異まで増幅するのに用いた。

In the first stage of PCR amplification, the first N-terminal and C-terminal fragments of construct 1966 were independently amplified. An N-terminal fragment was generated using primers EPO7 and EPO8. EPO7 incorporates Bam HI (shown in italics) and has a kozak sequence (shown underlined) followed by the first 18 nucleotides encoding the first 6 amino acids of the full-length EPO ORF. In the EPO8 primer, the codon CGA encoding the Arg residue at amino acid 140 (highlighted in bold) is changed to the codon GGA encoding the Gly residue. C-terminal fragments were generated using primers EPO9 and EPO10. In EPO9, the underlined sequence is the Cla I site, and the Cla I site and subsequent DNA are the reverse complements of the DNA encoding the first 10 amino acids of the mature HSA protein. In EPO9, the last 5 nucleotides correspond to the reverse complement of the last 5 nucleotides in full length EPO, which deletes the final Arg-193 residue. The EPO10 primer is 136-143 of the full-length form of EPO, except that the codon CGA encoding the Arg residue at amino acid 140 (highlighted in bold) is changed to the codon GGA encoding the Gly residue. Contains a nucleic acid sequence encoding an amino acid. In the second stage of PCR amplification, primers EPO7 and EPO9 contain full-length EPO with Arg-140 and the reaction mixture contained both PCR-amplified N-terminal fragment and PCR-amplified C-terminal fragment. Used to amplify up to mutation.

The PCR product was purified and subsequently digested with Bam HI and Cla I. And after further purification of the Bam HI-Cla I fragment by gel electrophoresis, this product was cloned into pC4: HSA digested with Bam HI / Cla I to give construct number 2294.
Furthermore, analysis of the N-terminus of albumin fusion protein by amino acid sequencing confirmed the presence of the predicted EPO sequence. (See below).

  The EPO albumin fusion protein of the invention is preferably an HSA fused to either the N-terminus or C-terminus of the mature form of EPO lacking the final Arg residue (ie, Ala-28 to Asp-192). Includes the mature form (ie Asp-25-Leu-609). In one embodiment of the invention, the EPO albumin fusion protein of the invention further comprises a signal sequence that directs the nascent fusion polypeptide in the secretory pathway of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed and the mature EPO albumin fusion protein is secreted directly into the culture medium. The EPO albumin fusion protein of the present invention includes heterologous signal sequences (MAF, INV, Ig, Fibulin B, Clusterin, insulin-like growth factor binding protein 4, modified HSA leader sequences (including chimeric HSA / MAF leader sequences, Or other heterologous signal sequences known in the art, including but not limited to). In a preferred embodiment, the EPO albumin fusion protein of the invention comprises a native EPO signal sequence. In a further preferred embodiment, the EPO albumin fusion protein of the present invention further comprises an N-terminal methionine residue. Polynucleotides (including fragments and / or variants) encoding these polypeptides are also encompassed by the present invention.

(Expression and purification of construct ID 2294)
(Expression in CHO cells)
Construct 2294 can be transfected into CHO cells as described in Examples 6 and 8. Expression levels and specific productivity rates can be determined as described in Example 8.

(Purification from CHO supernatant)
Cell supernatants containing EPO-HSA fusion protein expressed from construct ID # 2294 in CHO cells can be purified as in Examples 7 and 8. N-terminal sequencing should yield the sequence APPRLI (SEQ ID NO: 2141), which corresponds to the amino terminus of the mature form of EPO, and should yield a protein of about 87.7 kDa MW.

(In vitro TF-1 cell proliferation assay for construct 2294)
(Method)
The in vitro TF-1 cell proliferation assay for the EPO-HSA albumin fusion encoded by construct 2294 is described earlier in Example 8 under the subsection title “In vitro TF-1 cell proliferation assay for construct 1966”. Can be implemented as described.

(The activity of construct 2294 can be assayed using an in vivo Harlan mouse model for the measurement of hematocrit)
Using the in vivo Harlan mouse model as described above under the subsection “In Vivo Harlan Mouse Model for Measurement of Hematocrit” in Example 8, the EPO albumin fusion protein encoded by construct 2294 Hematocrit levels can be measured.

(Example 12: Construct ID 2298, EPO-HSA, production)
Construct ID 2298 (pEE12.1: EPO.R140G.HSA) contains DNA encoding an EPO albumin fusion protein, which is fused to the amino terminus of the mature form of HSA cloned into the mammalian expression vector pEE12.1. It has the full-length EPO protein (including the native leader sequence) except the last Arg residue fused (ie, M1-D192 with a point mutation that mutates Arg-140 to Gly).

(Cloning of EPO cDNA for construct 2298)
Construct ID # 2298 encodes an albumin fusion protein containing the leader sequence and mature form of EPO, followed by the mature HSA protein. Construct ID # 2298 was generated by using construct ID # 1997 (ie, pEE12.1: EPO.M1-D192.HSA) as a template for PCR mutagenesis.

  Two oligonucleotides (EPO11 and EPO12) suitable for PCR amplification of the template with construct ID # 1997 were synthesized.

ＥＰＯ１１アンチセンスプライマーは、全長形態のＥＰＯのアミノ酸１３５〜１４５にわたる配列の逆相補体を含む（アミノ酸１４０におけるＡｒｇをコードするコドンＣＧＡ（太字で強調されている）が、Ｇｌｙ残基をコードするコドンＧＧＡに変更されていることを例外とする）。ＥＰＯ１２センスプライマーは、全長形態のＥＰＯのアミノ酸１３５〜１４５をコードする核酸配列を含む（アミノ酸１４０におけるＡｒｇ残基をコードするコドンＣＧＡ（太字で強調されている）が、Ｇｌｙ残基をコードするコドンＧＧＡに変更されていることを例外とする）。部位特異的変異誘発キットおよびＳｔｒａｔａｇｅｎｅからのプロトコルを使用して、ＰＣＲ反応は、ＡｒｇからＧｌｙへの変異を除く、構築物ＩＤ ＃１９９７の全テンプレートを作製した。このＰＣＲ産物をＤｐｎ Ｉで消化し、コンピテントＸＬ１ Ｂｌｕｅ細菌に形質転換し、そしてコロニーを配列決定して、確認した。Ｄｐｎ Ｉエンドヌクレアーゼは、メチル化および半メチル化されたＤＮＡに対して特異的であり、そして配列５’−ＧｍＡＴＣ−３’を標的化する。Ｄｐｎ Ｉを使用して、親ＤＮＡテンプレートを消化し、変異含有合成ＤＮＡを選択する。

The EPO11 antisense primer contains the reverse complement of the sequence spanning amino acids 135-145 of the full-length form of EPO (codon CGA encoding Arg at amino acid 140 (highlighted in bold) but codon encoding the Gly residue) (The exception is GGA). The EPO12 sense primer includes a nucleic acid sequence encoding amino acids 135-145 of full-length EPO (codon CGA encoding Arg residue at amino acid 140 (highlighted in bold)), but codon encoding Gly residue. (The exception is GGA). Using the site-directed mutagenesis kit and the protocol from Stratagene, the PCR reaction generated an entire template with construct ID # 1997, excluding the Arg to Gly mutation. The PCR product was digested with Dpn I, transformed into competent XL1 Blue bacteria, and the colonies were sequenced and confirmed. Dpn I endonuclease is specific for methylated and hemimethylated DNA and targets the sequence 5′-GmATC-3 ′. Dpn I is used to digest the parent DNA template and select the mutation-containing synthetic DNA.

  Furthermore, analysis by amino acid sequencing of the N-terminus of the expressed albumin fusion protein can confirm the presence of the predicted EPO sequence (see below).

  The EPO albumin fusion protein of the present invention is preferably a mature HSA fused to either the N-terminus or C-terminus of the mature form of EPO lacking the last Arg residue (ie, Ala-28 to Asp-192). Including the form (ie Asp-25 to Leu-609). In one embodiment of the invention, the EPO albumin fusion protein of the invention further comprises a signal sequence that directs the nascent fusion polypeptide in the secretory pathway of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed and the mature EPO albumin fusion protein is secreted directly into the culture medium. The EPO albumin fusion protein of the present invention includes heterologous signal sequences (MAF, INV, Ig, Fibulin B, Clusterin, insulin-like growth factor binding protein 4, modified HSA leader sequences (including chimeric HSA / MAF leader sequences, Or other heterologous signal sequences known in the art, including but not limited to). In a preferred embodiment, the EPO albumin fusion protein of the invention comprises a native EPO signal sequence. In a further preferred embodiment, the EPO albumin fusion protein of the present invention further comprises an N-terminal methionine residue. Polynucleotides (including fragments and / or variants) encoding these polypeptides are also encompassed by the present invention.

(Expression and purification of construct ID 2298)
(Expression in NS0 cells)
Construct 2298 can be transfected into NS0 cells as described in Examples 6 and 10. Expression levels and specific production rates can be determined as described in Example 8.

(Purification from NS0 cell supernatant)
Cell supernatants containing EPO-HSA fusion protein expressed from construct ID # 2298 in NS0 cells can be purified as in Examples 7 and 10. N-terminal sequencing should yield the sequence APPRLI (SEQ ID NO: 2141), which corresponds to the amino terminus of the mature form of EPO, and should yield a protein of about 87.7 kDa MW.

(In vitro TF-1 cell proliferation assay for construct 2298)
(Method)
The in vitro TF-1 cell proliferation assay for the EPO-HSA albumin fusion protein encoded by construct 2298 is described in Example 8 in the subsection titled “In vitro TF-1 cell proliferation assay for the albumin fusion protein encoded by construct 1966”. And under the subsection title “In Vitro TF-1 Cell Proliferation Assay for Construct 1997” in Example 10 as described above.

(The activity of construct 2298 can be assayed using an in vivo Harlan mouse model for measurement of hematocrit)
The EPO encoded by construct 2298 using the in vivo Harlan mouse model as described above in Example 8 under the subheading “In Vivo Harlan Mouse Model for Measurement of Hematocrit” and above in Example 10. The hematocrit level of the albumin fusion protein can be measured.

(Example 13: Construct ID 2325, EPO-HSA, production)
The optimized construct ID 2325 (pC4.EPO: M1-D192.HSA) codon contains DNA encoding the EPO albumin fusion protein, which is a mature form of HSA cloned into the mammalian expression vector pC4. It has a full-length EPO protein (including the native leader sequence) fused to the amino terminus (ie, M1-D192 with a point mutation from Arg-140 to Gly).

(Cloning of EPO cDNA for construct 2325)
The DNA encoding the EPO open reading frame was a codon optimized to not hybridize to the wild type EPO gene sequence. A polynucleotide encoding EPO was PCR generated with 6 overlapping oligonucleotides and cloned into a TA vector. Construct ID # 2325 encodes an albumin fusion protein comprising a leader sequence and a mature form of EPO followed by a mature HSA protein.

  Furthermore, analysis by amino acid sequencing of the N-terminus of the expressed albumin fusion protein can confirm the presence of the predicted EPO sequence (see below).

  The EPO albumin fusion protein of the present invention is preferably a mature HSA fused to either the N-terminus or C-terminus of the mature form of EPO lacking the last Arg residue (ie, Ala-28 to Asp-192). Including the form (ie Asp-25 to Leu-609). In one embodiment of the invention, the EPO albumin fusion protein of the invention further comprises a signal sequence that directs the nascent fusion polypeptide in the secretory pathway of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed and the mature EPO albumin fusion protein is secreted directly into the culture medium. The EPO albumin fusion protein of the present invention includes heterologous signal sequences (MAF, INV, Ig, Fibulin B, Clusterin, insulin-like growth factor binding protein 4, modified HSA leader sequences (including chimeric HSA / MAF leader sequences, Or other heterologous signal sequences known in the art, including but not limited to). In a preferred embodiment, the EPO albumin fusion protein of the invention comprises a native EPO signal sequence. In a further preferred embodiment, the EPO albumin fusion protein of the present invention further comprises an N-terminal methionine residue. Polynucleotides (including fragments and / or variants) encoding these polypeptides are also encompassed by the present invention.

(Expression and purification of construct ID2325)
(Expression in CHO cells)
Construct 2325 can be transfected into CHO cells as described in Examples 6 and 8. Expression levels and specific production rates can be determined as described in Example 8.

(Purification from CHO supernatant)
Cell supernatants containing EPO-HSA fusion protein expressed from construct ID # 2325 in CHO cells can be purified by the methods described in Examples 7 and 8. N-terminal sequencing should yield the sequence APPRLI (SEQ ID NO: 2141), which corresponds to the amino terminus of the mature form of EPO, and should yield a protein of about 87.7 kDa MW.

(In vitro TF-1 cell proliferation assay for construct 2325)
(Method)
The in vitro TF-1 cell proliferation assay for the EPO-HSA albumin fusion encoded by construct 2325 is described earlier in Example 8 under the subsection title “In vitro TF-1 cell proliferation assay for construct 1966”. Can be implemented as described.

(The activity of construct 2325 can be assayed using an in vivo Harlan mouse model for measurement of hematocrit)
Using the in vivo Harlan mouse model as described above under the subsection title “In Vivo Harlan Mouse Model for Measurement of Hematocrit” in Example 8, the EPO albumin fusion protein encoded by construct 2325 Hematocrit levels can be measured.

(Example 14: EPO albumin fusion protein indication)
The results from the above in vitro and in vivo assays show that EPO albumin fusion protein is in various states (end stage renal disease (dialysis patients), chronic renal failure before dialysis, zidovudine-treated HIV patients, cancer patients undergoing chemotherapy And can be used in the treatment of bleeding disorders and anemia caused by, but not limited to, premature babies). EPO albumin fusion proteins can also be used to reduce the need for blood transfusions prior to surgery in anemic patients undergoing selective non-cardiac non-vascular surgery. Indications in the development of these drugs include: aplastic and other refractory anemias, refractory anemia in inflammatory bowel disease, and transfusion escape in selective orthopedic surgery. Renal disease and anemia in tumors are the two main indications for the EPO albumin fusion protein encoded by constructs 1966, 1981, 1997, 2294, 2298 and 2325.

(Example 15: Construct ID 1812, IL2-HSA, production)
Construct ID 1812 (pSAC35: IL2.A21-T153.HSA) contains DNA encoding the IL2 albumin fusion protein, which is the yeast S. cerevisiae. It has the HSA chimeric leader sequence (ie HSA-kex2 signal peptide), mature IL2 protein form (ie A21-T153) fused to the amino terminus of the mature form of HSA in the cerevisiae expression vector pSAC35.

(Cloning of IL2 cDNA)
The polynucleotide encoding IL2 was PCR amplified using primers IL2-1 and IL2-2 as described below. These amplifiers were cut with Sal I / Cla I and ligated to pScCHSA cut with Xho I / Cla I. Construct ID # 1812 encodes an albumin fusion protein comprising the chimeric leader sequence of HSA, the mature form of IL2, followed by the mature HSA protein.

  Two oligonucleotides (IL2-1 and IL2-2) suitable for PCR amplification of the polynucleotide encoding the mature form of IL2 were synthesized:

ＩＬ２−１は、Ｓａｌ Ｉクローニング部位（下線を引いて示される）、ＨＳＡキメラリーダー配列の最後の３アミノ酸残基をコードするヌクレオチド、およびＩＬ２の成熟形態の最初の８アミノ酸残基をコードする２４ヌクレオチドを組み込む。ＩＬ２−２において、Ｃｌａ Ｉ部位（下線を引いて示される）およびそれに続くＤＮＡは、成熟ＨＳＡタンパク質（配列番号１０３８）の最初の１０個のアミノ酸をコードするＤＮＡの逆相補体であり、そして最後の２４個のヌクレオチドは、ＩＬ２の最後の８個のアミノ酸残基をコードするＤＮＡの逆相補体である（実施例２を参照のこと）。ＩＬ２−ＨＳＡのＰＣＲ増幅体を、これらのプライマーを使用して産生し、精製し、Ｓａｌ Ｉ制限酵素およびＣｌａ Ｉ制限酵素で消化し、そしてｐＳｃＣＨＳＡベクターのＸｈｏ Ｉ部位およびＣｌａ Ｉ部位にクローニングした。配列を確認した後に、このＩＬ２アルブミン融合タンパク質をコードする発現カセットを、Ｎｏｔ Ｉフラグメントとして、ｐＳＡＣ３５にサブクローニングした。

IL2-1 encodes the Sal I cloning site (shown underlined), the nucleotide encoding the last 3 amino acid residues of the HSA chimeric leader sequence, and the first 8 amino acid residues of the mature form of IL2 24 Incorporates nucleotides. In IL2-2, the Cla I site (shown underlined) and subsequent DNA is the reverse complement of the DNA encoding the first 10 amino acids of the mature HSA protein (SEQ ID NO: 1038), and finally The 24 nucleotides of are reverse complements of DNA encoding the last 8 amino acid residues of IL2 (see Example 2). PCR amplifications of IL2-HSA were produced using these primers, purified, digested with Sal I and Cla I restriction enzymes, and cloned into the Xho I and Cla I sites of the pScCHSA vector. After confirming the sequence, the expression cassette encoding this IL2 albumin fusion protein was subcloned into pSAC35 as a Not I fragment.

  Furthermore, analysis by amino acid sequencing of the N-terminus of the expressed albumin fusion protein can confirm the presence of the predicted IL2 sequence (see below).

  The IL2 albumin fusion protein of the invention is preferably a mature form of HSA (ie Asp-25 to 25) fused to either the N-terminus or C-terminus of the mature form of IL2 (ie Ala-21 to Thr-153). Leu-609). In one embodiment of the invention, the IL2 albumin fusion protein of the invention further comprises a signal sequence that directs the nascent fusion polypeptide in the secretory pathway of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed and the mature IL2 albumin fusion protein is secreted directly into the culture medium. The IL2 albumin fusion protein of the present invention includes heterologous signal sequences (MAF, INV, Ig, Fibulin B, Clusterin, insulin-like growth factor binding protein 4, modified HSA leader sequences (including chimeric HSA / MAF leader sequences, Or other heterologous signal sequences known in the art, including but not limited to). In a preferred embodiment, the IL2 albumin fusion protein of the invention comprises a native IL2 signal sequence. In a further preferred embodiment, the IL2 albumin fusion protein of the invention further comprises an N-terminal methionine residue. Polynucleotides (including fragments and / or variants) encoding these polypeptides are also encompassed by the present invention.

(Expression and purification of construct ID 1812)
(Expression in yeast S. cerevisiae)
Construct 1812, yeast S. cerevisiae. Transfection into cerevisiae strain BXP10 was performed by methods known in the art (see Example 3), and cells were harvested at steady state 72 hours after growth. Supernatant from yeast transfected with construct 1812 was collected by cleaning the cells at 3000 g for 10 minutes.
Expression levels were tested by immunoblot detection using anti-HSA serum (Kent Laboratories) as the primary antibody. An IL2 albumin fusion protein with a molecular weight of about 85 kDa was obtained. Specific production rates were determined by ELISA, where the capture antibody was a US Biological # A1327-53 monoclonal anti-HSA antibody or a monoclonal anti-human IL2 antibody (eg, Biosource # AHCO422, Pharmingen # 555051, R & D Systems # MAB202, or R & D And the detection antibody is a monoclonal anti-human IL2-biotinylated antibody (eg, derived from Biosource # AHC069 or Endogen / Pierce # M-600-B), or a monoclonal anti-HSA antibody (Biotrend #, respectively). 4T24) and the conjugate was horseradish peroxidase / streptavidin (Vector Laborato). ies, a # SA-5004), and substrates were KPL TMB peroxidase substrate (KPL # 50-76-01). Analysis was performed according to manufacturer's protocol and / or by methods known in the art.

(Purification from the supernatant of yeast S. cerevisiae cells)
Yeast S. Cell supernatants containing IL2 albumin fusion protein expressed from construct ID # 1812 in cerevisiae cells, either on a small scale with a Dyax peptide affinity column (see Example 4), or on a large scale with the following five steps: Purified with: Diafiltration, Anion Exchange Chromatography using DEAE-Sepharose Fast Flow column, Hydrophobic Interaction Chromatography (HIC) using Butyl 650S column, using SP-Sepharose Fast Flow column High performance chromatograph using cation exchange chromatography or Blue-Sepharose chromatography, and Q-Sepharose high performance column chromatography I by chromatography (see Example 4). The IL2 albumin fusion protein is eluted from the DEAE-Sepharose Fast Flow column with 100-250 mM NaCl, eluted from the SP-Sepharose Fast Flow column with 150-250 mM NaCl, and from the Q-Sepharose High Performance column, Elution was performed at 5 to 7.5 mS / cm. N-terminal sequencing should yield the sequence APTSSST corresponding to the amino terminus of the mature form of IL2.

(IL2 activity can be assayed using in vitro T and NK cell line proliferation assays)
The mouse CTLL T cell line is used, which is completely dependent on IL2 for cell proliferation and survival. This cell line expresses high levels of high affinity IL2 receptor and is very sensitive to very low doses of IL2.

(Method)
CTLL-2 cells (mouse IL2-dependent T cell line) are grown in RPMI 10% FBS containing 5 ng / mL recombinant IL2 and BME. Prior to the assay, these cells are washed twice with PBS to remove IL2. 1 × 10 ⁴ cells / well were seeded in 96 well plates with a final volume of 200 μl of RPMI 10% FBS. Yeast and 293T supernatants are tested at final concentrations of 10%, 5% and 1%. In addition, recombinant human IL2, “hfIL2”, is diluted in the negative control supernatant (HSA only) to test the effect of media on recombinant protein stability. These cells are cultured at 37 ° C. for 20 hours and then pulsed with 1 μCi of ³ H-thymidine for 6 hours. Proliferation is measured by thymidine incorporation and each sample is tested in triplicate.

(The activity of the IL2 albumin fusion protein encoded by construct 1812 can be assayed using in vitro T and NK cell line proliferation assays)
(Method)
CTLL-2 cells (mouse IL2-dependent T cell line) were grown in RPMI 10% FBS containing 5 ng / mL recombinant IL2 and BME. Prior to the assay, these cells were washed twice with PBS to remove IL2. 1 × 10 ⁴ cells / well were seeded in 96 well plates with a final volume of 200 μl of RPMI 10% FBS. Yeast and 293T supernatants were tested at final concentrations of 10%, 5% and 1%. In addition, recombinant human IL2, “rhIL2”, was diluted in the negative control supernatant (HSA only) to test the effect of media on the stability of the recombinant protein. These cells were cultured at 37 ° C. for 20 hours and then palmed with 1 μCi of ³ H-thymidine for 6 hours. Proliferation was measured by thymidine incorporation and each sample was tested in triplicate.

(result)
IL2 albumin fusion construct ID # 1812 stimulated proliferation of CTLL-2 cells in a dose-dependent manner (see FIG. 9).

(The activity of the IL2 albumin fusion protein encoded by construct 1812 can be assayed using an in vivo BALB / c model: RENCA tumor response to therapy)
The mouse model uses BALB / c mouse RENCA adenocarcinoma. The RENCA tumor used in these studies occurred naturally. This RENCA tumor originally originated from Dr. NCI (Bethesda, MD). Isolated by Sarah Stewart. RENCA tumors grow gradually after the transfer of as few as 50 viable cells and spontaneously metastasize from intrarenal transplants to local lymph nodes, lungs, liver and spleen, and other organs. The immunogenicity of RENCA has been determined to be low to moderate. Mice bearing RENCA usually die within 35-40 days after intrarenal injection of 1 × 10 ⁵ RENCA tumor cells. Mice given intraperitoneally with the same number of RENCA tumor cells usually die within 30-50 days.

(Method)
The abdomen in the BALB / c mice (6-8 weeks old) (n = 10), 10 ⁵ RENCA cells obtained from in vivo fourth passage were injected subcutaneously. Daily (QD) or every other day with placebo (PBS), HSA, rhIL2 (0.122 mg / kg / QD or 200,000 or 300,000 U / mouse), or IL2 albumin fusion protein (0.61 mg / kg) Ten days after (QOD), mice were monitored for changes in tumor size at day 14, 17, 21, 25, 28 or 31 after tumor seeding. Data is represented in a dot analysis, where each dot represents a single animal. The horizontal line in each group represents the average value (see FIG. 10).

(result)
The IL2 albumin fusion protein encoded by construct ID # 1812 was tested in the above assay.

  Administration of IL2 albumin fusion protein expressed from construct ID # 1812 daily or every other day had a significant effect on tumor growth resulting in delayed growth and / or reduced tumor size. Administration every other day was more advantageous. This is because the tolerance level was higher (see FIG. 10). From the day of seeding to day 31, 3 out of 10 mice that received IL2 albumin fusion product had no tumor, only 2 showed signs of reduced tumor, and Four mice had small tumors that were thought to have contracted. Only one mouse did not respond favorably to this treatment. Daily treatment with IL2 albumin fusion protein also resulted in delayed growth or actual contraction of the tumor (2 out of 10 were tumor free and the remaining 7 mice at the end of the experiment) Had small tumors and two had large tumors). All animals that received 0.61 mg / kg IL2 albumin fusion protein were alive on the end day, while only 40% of mice that received placebo (PBS) and HSA 70% of the mice were alive. Biological effects were shown to be much greater than daily administration of either 200,000 U / mouse or 300,000 U / mouse of recombinant human IL2. Recombinant human IL2 had a poor effect on tumor growth (all mice receiving rhIL2 developed tumors and the only effect observed was growth delay). Of the 10 mice that received rhIL2 (200,000 or 300,000 U / mL), 3 died by day 31. The low dose of 0.122 mg / kg / day tested did not inhibit tumor growth or help mice from tumor-related death. The IL2 albumin fusion protein potently inhibited in vivo RENCA growth and in some cases resulted in complete recovery from the tumor.

(Example 16: Construct ID 2030, IL2-HSA, production)
Construct ID 2030 (pSAC35: ycoIL2.A21-T153.HSA) contains DNA encoding the IL2 albumin fusion protein, which is the yeast S. cerevisiae. It has the HSA chimeric leader sequence (ie HSA-kex2 signal peptide), the mature form of the IL2 protein (ie A21-T153) fused to the amino terminus of the mature form of HSA in the cerevisiae expression vector pSAC35.

(Cloning of IL2 cDNA)
The IL2 open reading frame “ORF” DNA was a codon optimized to not hybridize to the wild type IL2 gene. A polynucleotide encoding codon optimized IL2 was PCR generated with 6 overlapping oligonucleotides and cloned into a TA vector. The polynucleotide encoding this codon optimized IL2 was PCR amplified from this clone using primers IL2-3 and IL2-4 described below, cut with Sal I / Cla I, and Xho I / Cla I Ligated into pScCHSA cut with I. Construct ID # 2030 encodes an albumin fusion protein comprising a chimeric leader sequence of HSA and a mature form of IL2 fused to the amino terminus of the mature form of HSA.

  Two oligonucleotides (IL2-3 and IL2-4) suitable for PCR amplification of codon-optimized polynucleotides encoding the mature form of IL2 were synthesized.

ＩＬ２−３は、Ｓａｌ Ｉクローニング部位（下線を引いて示される）、ＨＳＡキメラリーダー配列の最後の３アミノ酸残基をコードするヌクレオチド、およびＩＬ２の成熟形態の最初の８アミノ酸残基をコードする２４ヌクレオチドを含む。ＩＬ２−４において、Ｃｌａ Ｉ部位（下線を引いて示される）およびそれに続くＤＮＡは、成熟ＨＳＡタンパク質（配列番号１０３８）の最初の１０個のアミノ酸をコードするＤＮＡの逆相補体であり、そして最後の２４個のヌクレオチドは、ＩＬ２の最後の８個のアミノ酸残基をコードするＤＮＡの逆相補体である（実施例２を参照のこと）。ＰＣＲ増幅体を、３つのプライマーを使用して産生し、精製し、Ｓａｌ Ｉ制限酵素およびＣｌａ Ｉ制限酵素で消化し、そしてｐＳｃＣＨＳＡベクターのＸｈｏ Ｉ部位およびＣｌａ Ｉ部位にクローニングした。配列を確認した後に、このＮｏｔ Ｉフラグメント含有ＩＬ２ｂアルブミン融合タンパク質発現カセットを、Ｎｏｔ Ｉで切断されるｐＳＡＣ３５にサブクローニングした。

IL2-3 encodes the Sal I cloning site (shown underlined), the nucleotide encoding the last 3 amino acid residues of the HSA chimeric leader sequence, and the first 8 amino acid residues of the mature form of IL2 24 Contains nucleotides. In IL2-4, the Cla I site (shown underlined) and subsequent DNA is the reverse complement of the DNA encoding the first 10 amino acids of the mature HSA protein (SEQ ID NO: 1038), and finally The 24 nucleotides of are reverse complements of DNA encoding the last 8 amino acid residues of IL2 (see Example 2). PCR amplifications were produced using three primers, purified, digested with Sal I and Cla I restriction enzymes, and cloned into the Xho I and Cla I sites of the pScCHSA vector. After confirming the sequence, this Not I fragment-containing IL2b albumin fusion protein expression cassette was subcloned into pSAC35 which is cleaved with Not I.

  Furthermore, analysis by amino acid sequencing of the N-terminus of the expressed albumin fusion protein can confirm the presence of the predicted IL2 sequence (see below).

  The IL2 albumin fusion protein of the invention is preferably a mature form of HSA (ie Asp-25 to 25) fused to either the N-terminus or C-terminus of the mature form of IL2 (ie Ala-21 to Thr-153). Leu-609). In one embodiment of the invention, the IL2 albumin fusion protein of the invention further comprises a signal sequence that directs the nascent fusion polypeptide in the secretory pathway of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed and the mature IL2 albumin fusion protein is secreted directly into the culture medium. The IL2 albumin fusion protein of the present invention includes heterologous signal sequences (MAF, INV, Ig, Fibulin B, Clusterin, insulin-like growth factor binding protein 4, modified HSA leader sequences (including chimeric HSA / MAF leader sequences, Or other heterologous signal sequences known in the art, including but not limited to). In a preferred embodiment, the IL2 albumin fusion protein of the invention comprises a native IL2 signal sequence. In a further preferred embodiment, the IL2 albumin fusion protein of the invention further comprises an N-terminal methionine residue. Polynucleotides (including fragments and / or variants) encoding these polypeptides are also encompassed by the present invention.

(Expression and purification of construct 2030)
(Expression in yeast S. cerevisiae)
Yeast S. C. cerevisiae strain BXP10 can be transfected by methods known in the art (see Example 3) as described previously for construct ID 1812 (see Example 15). .

(Purification from the supernatant of yeast S. cerevisiae cells)
Yeast S. Cell supernatant containing IL2-HSA expressed from construct ID # 2030 in cerevisiae cells, either on a small scale with a Dyax peptide affinity column (see Example 4) or on a large scale with the following five steps Can be purified by: Diafiltration, anion exchange chromatography using DEAE-Sepharose Fast Flow column, hydrophobic interaction chromatography (HIC) using Butyl 650S column, using SP-Sepharose Fast Flow column High performance chromatography using cation exchange chromatography or Blue-Sepharose chromatography and Q-Sepharose high performance column chromatography (Examples) And see Example 15). N-terminal sequencing should yield the sequence APTSSST (SEQ ID NO: 2142) corresponding to the amino terminus of the mature form of IL2.

(The activity of the IL2 albumin fusion protein encoded by construct 2030 can be assayed using in vitro T and NK cell line proliferation assays)
The activity of Construct ID 2030 can be assayed using in vitro T and NK cell line proliferation assays as in Example 15.

(The activity of IL2 albumin fusion protein encoded by construct 2030 can be assayed using an in vivo BALB / c model: RENCA tumor response to therapy)
The activity of the IL2 albumin fusion protein encoded by construct 2030 can be assayed using an in vivo BALB / c model as described in Example 15, where the RENCA tumor response to therapy is monitored.

(Example 17: Construct ID 2031, HSA-IL2, production)
Construct ID 2031 (pSAC35: HSA.ycoIL2.A21-T153) contains DNA encoding the IL2 albumin fusion protein, which is the yeast S. cerevisiae. It has the full-length HSA sequence containing the HSA chimeric leader sequence (ie, HSA-kex2 signal peptide) fused to the amino terminus of the mature form of IL2 (A21-T153) in the cerevisiae expression vector pSAC35.

(Cloning of IL2 cDNA)
The IL2 open reading frame “ORF” DNA was a codon optimized to not hybridize to the wild type IL2 gene. A polynucleotide encoding codon optimized IL2 was PCR generated with 6 overlapping oligonucleotides and cloned into a TA vector. The polynucleotide encoding this codon optimized IL2 is PCR amplified from this codon using primers IL2-5 and IL2-6 described below, cut with Bsu 36I / Pme I, and Bsu 36I / Pme I Ligated into pScNHSA cut with I. Construct ID # 2031 encodes an albumin fusion protein comprising a chimeric leader sequence and mature form of HSA, and a mature form of IL2.

  Two oligonucleotides (IL2-5 and IL2-6) were synthesized that were suitable for PCR amplification of the optimized codons of the polynucleotide encoding the mature form of IL2.

ＩＬ２−５は、Ｂｓｕ ３６Ｉクローニング部位（下線を引いて示される）、ＨＳＡの成熟形態の最後の４アミノ酸残基をコードするヌクレオチド、およびＩＬ２の成熟形態の最初の８アミノ酸残基をコードする２４ヌクレオチドを含む。ＩＬ２−６において、Ｐｍｅ Ｉ部位は、下線を引いて示され（配列番号８３４）、そして最後の２４ヌクレオチドは、ＩＬ２の最後の８アミノ酸残基をコードするＤＮＡの逆相補体である（実施例２を参照のこと）。ＰＣＲ増幅体を、これらのプライマーを使用して産生し、精製し、Ｂｓｕ ３６Ｉ制限酵素およびＰｍｅ Ｉ制限酵素で消化し、そしてｐＳｃＮＨＳＡベクターのＢｓｕ ３６Ｉ部位およびＰｍｅ Ｉ部位にクローニングした。配列を確認した後に、このＩＬ２アルブミン融合タンパク質発現カセットを含むＮｏｔ Ｉフラグメントを、Ｎｏｔ Ｉで切断したｐＳＡＣ３５にサブクローニングした。

IL2-5 encodes the Bsu 36I cloning site (shown underlined), the nucleotide encoding the last 4 amino acid residues of the mature form of HSA, and the 24 encoding the first 8 amino acid residues of the mature form of IL2. Contains nucleotides. In IL2-6, the Pme I site is shown underlined (SEQ ID NO: 834) and the last 24 nucleotides are the reverse complement of DNA encoding the last 8 amino acid residues of IL2 (Examples). 2). PCR amplifications were produced using these primers, purified, digested with Bsu 36I and Pme I restriction enzymes, and cloned into the Bsu 36I and Pme I sites of the pScNHSA vector. After confirming the sequence, the Not I fragment containing this IL2 albumin fusion protein expression cassette was subcloned into pSAC35 cut with Not I.

  Furthermore, analysis by amino acid sequencing of the N-terminus of the expressed albumin fusion protein can confirm the presence of the predicted HSA sequence (see below).

  The IL2 albumin fusion protein of the invention is preferably a mature form of HSA (ie Asp-25 to 25) fused to either the N-terminus or C-terminus of the mature form of IL2 (ie Ala-21 to Thr-153). Leu-609). In one embodiment of the invention, the IL2 albumin fusion protein of the invention further comprises a signal sequence that directs the nascent fusion polypeptide in the secretory pathway of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed and the mature IL2 albumin fusion protein is secreted directly into the culture medium. The IL2 albumin fusion protein of the present invention includes heterologous signal sequences (MAF, INV, Ig, Fibulin B, Clusterin, insulin-like growth factor binding protein 4, modified HSA leader sequences (including chimeric HSA / MAF leader sequences, Or other heterologous signal sequences known in the art, including but not limited to). In a preferred embodiment, the IL2 albumin fusion protein of the invention comprises a native IL2 signal sequence. In a further preferred embodiment, the IL2 albumin fusion protein of the invention further comprises an N-terminal methionine residue. Polynucleotides (including fragments and / or variants) encoding these polypeptides are also encompassed by the present invention.

(Expression and purification of construct ID 2031)
(Expression in yeast S. cerevisiae)
Yeast S. C. cerevisiae strain BXP10 can be transfected by methods known in the art (see Example 3) as described previously for construct ID 1812 (see Example 15). .

(Purification from the supernatant of yeast S. cerevisiae cells)
Yeast S. Cell supernatants containing HSA-IL2 expressed from construct ID # 2031 in cerevisiae cells, either on a small scale with a Dyax peptide affinity column (see Example 4) or on a large scale with the following five steps Can be purified by: Diafiltration, anion exchange chromatography using DEAE-Sepharose Fast Flow column, hydrophobic interaction chromatography (HIC) using Butyl 650S column, using SP-Sepharose Fast Flow column High performance chromatography using cation exchange chromatography or Blue-Sepharose chromatography and Q-Sepharose high performance column chromatography (Examples) And see Example 15). N-terminal sequencing should yield the sequence DAHKS (SEQ ID NO: 2143) corresponding to the amino terminus of the mature form of HSA.

(The activity of the IL2 albumin fusion protein encoded by construct 2031 can be assayed using in vitro T and NK cell line proliferation assays)
The activity of Construct ID 2031 can be assayed using in vitro T and NK cell line proliferation assays as described in Example 15.

(The activity of the IL2 albumin fusion protein encoded by construct 2031 can be assayed using an in vivo BALB / c model: RENCA tumor response to therapy)
The activity of the IL2 albumin fusion protein encoded by construct 2031 can be assayed using an in vivo BALB / c model as described in Example 15, where the RENCA tumor response to therapy is monitored.

(Example 18: Indication for IL2 albumin fusion protein)
Indications of IL2 albumin fusion proteins (including but not limited to those encoded by constructs 1812, 2030 and 2031) include, but are not limited to: solid tumors, metastatic kidney cells Carcinoma, metastatic melanoma, malignant melanoma, renal cell carcinoma, treatment of HIV infection (AIDS), inflammatory bowel disorder, capage sarcoma, leukemia, multiple sclerosis, rheumatoid arthritis, transplant rejection, type I diabetes, lung cancer Acute myeloid leukemia, hepatitis C, non-Hodgkin lymphoma, and ovarian cancer.

(Example 19: Construct ID 1642, GCSF-HSA, production)
Construct ID 1642 (pSAC35: GCSF.T31-P204.HSA) contains DNA encoding the GCSF albumin fusion protein, which is the yeast S. cerevisiae. HSA chimeric leader sequence (ie HSA-kex2 signal peptide), granulocyte colony stimulating factor, “G-CSF” protein (ie T31-P204) fused to the amino terminus of the mature form of HSA in the cerevisiae expression vector pSAC35 The “short form” of the mature form.

(GCSF cDNA cloning)
The polynucleotide encoding GCSF was PCR amplified using the primers GCSF-1 and GCSF-2 described below. The amplified was cut with Sal I / Cla I and ligated to pScCHSA cut with Xho I / Cla I. Construct ID # 1642 contains the DNA encoding the albumin fusion protein, including the chimeric leader sequence of HSA, the mature form of GCSF, followed by the mature HSA protein.

  Two oligonucleotides (GCSF-1 and GCSF-2) suitable for PCR amplification of the polynucleotide encoding the mature form of GCSF were synthesized:

ＧＣＳＦ−１は、Ｓａｌ Ｉクローニング部位（下線を引いて示される）、ＨＳＡキメラリーダー配列の最後の３アミノ酸残基をコードするヌクレオチド、およびＧＣＳＦの成熟形態の最初の６アミノ酸残基をコードする２０ヌクレオチドを含む。ＧＣＳＦ−２において、Ｃｌａ Ｉ部位（下線を引いて示される）およびそれに続くＤＮＡは、成熟ＨＳＡタンパク質（配列番号１０３８）の最初の１０個のアミノ酸をコードするＤＮＡの逆相補体であり、そして最後の２１個のヌクレオチドは、ＧＣＳＦの最後の７個のアミノ酸残基をコードするＤＮＡの逆相補体である。ＰＣＲ増幅体（ａｍｐｌｉｍｅｒ）を、これらのプライマーを使用して産生し、精製し、Ｓａｌ Ｉ制限酵素およびＣｌａ Ｉ制限酵素で消化し、そしてｐＳｃＣＨＳＡベクターのＸｈｏ Ｉ部位およびＣｌａ Ｉ部位にクローニングした。配列を確認した後に、このＧＣＳＦアルブミン融合発現カセットを含むＮｏｔ Ｉフラグメントを、Ｎｏｔ Ｉで切断されたｐＳＡＣ３５にサブクローニングした。

GCSF-1 encodes the Sal I cloning site (shown underlined), the nucleotide encoding the last 3 amino acid residues of the HSA chimeric leader sequence, and the first 6 amino acid residues of the mature form of GCSF 20 Contains nucleotides. In GCSF-2, the Cla I site (shown underlined) and subsequent DNA is the reverse complement of the DNA encoding the first 10 amino acids of the mature HSA protein (SEQ ID NO: 1038), and finally These 21 nucleotides are the reverse complement of DNA encoding the last 7 amino acid residues of GCSF. PCR amplifiers were produced using these primers, purified, digested with Sal I and Cla I restriction enzymes, and cloned into the Xho I and Cla I sites of the pScCHSA vector. After confirming the sequence, the Not I fragment containing this GCSF albumin fusion expression cassette was subcloned into pSAC35 cut with Not I.

  Furthermore, analysis by amino acid sequencing of the N-terminus of the expressed albumin fusion protein confirmed the presence of the predicted GCSF sequence (see below).

  The GCSF albumin fusion protein of the present invention is preferably a mature form of HSA (ie Asp-25 to 25) fused to either the N-terminus or C-terminus of the mature form of GCSF (ie Thr-31 to Pro-204). Leu-609). In one embodiment of the invention, the GCSF albumin fusion protein of the invention further comprises a signal sequence that directs the nascent fusion polypeptide in the secretory pathway of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed and the mature GCSF albumin fusion protein is secreted directly into the culture medium. The GCSF albumin fusion protein of the present invention includes heterologous signal sequences (MAF, INV, Ig, Fibulin B, Clusterin, insulin-like growth factor binding protein 4, modified HSA leader sequences (including chimeric HSA / MAF leader sequences, Or other heterologous signal sequences known in the art, including but not limited to). In a preferred embodiment, the GCSF albumin fusion protein of the invention comprises a native GCSF signal sequence. In a further preferred embodiment, the GCSF albumin fusion protein of the present invention further comprises an N-terminal methionine residue. Polynucleotides (including fragments and / or variants) encoding these polypeptides are also encompassed by the present invention.

(Expression and purification of construct ID 1642)
(Expression in yeast S. cerevisiae)
Construct 1642, yeast S. p. Transformation into cerevisiae strains D88, BXP10 and DXY1-aYAP3 mutants was performed by methods known in the art (see Example 3). When this transformed yeast produced the protein encoded by the fusion construct, a preliminary “halo assay” was performed. Secretion of the HSA fusion protein into an agar medium containing anti-HSA antibodies results in the formation of an insoluble “precipitate” ring or halo. The size of this halo is proportional to the amount of HSA protein produced. LEU2 + prototrophs were selected on synthetic complete leucine dropout medium (“SCD-Leu”) containing dextrose. Selected colonies and positive controls were gridded on BMMD plates containing anti-HSA antibodies. After growth, these plates were incubated at 4 ° C. to form precipitin rings. Based on the “halo assay”, colonies from transformation of construct 1642 produced protein. To establish the degree of secretion, transformed cells were harvested in stationary phase after being grown in suspension for 48 hours. The supernatant was collected by removing cells at 3000g for 10 minutes. Expression levels were tested by immunoblot detection using antibodies against anti-HSA serum (Kent Laboratories) or the therapeutic protein portion of the albumin fusion protein (ie, GCSF). A GCSF albumin fusion protein with a molecular weight of about 88 kDa was obtained. To obtain a workable quantity for purification, yeast transformants were seeded at 29.5 ° C. at 150 rpm in 1 L of BMM medium. The culture was centrifuged and passed through a 0.45 m filter. The specific production rate is determined by ELISA, where, for example, the capture antibody is R & D Systems Clone 3316.111 monoclonal mouse anti-GCSF and the detection antibody is R & D Systems BAF214 (ie, Clone ACN030081) biotinylated goat anti-human. A GCSF antibody, the conjugate is horseradish peroxidase / streptavidin (Vector Laboratories, # SA-5004) and the substrate is KPL TMB Peroxidase Substrate (KPL # 50-76-01), where This analysis is performed according to the manufacturer's protocol and / or by methods known in the art.

(Purification from the supernatant of yeast S. cerevisiae cells)
The general purification procedure for albumin fusion protein is described in Example 4. Purification of the GCSF albumin fusion protein is described in detail below. Another purification scheme is described in Example 20.

(Step 1: Phenyl high-speed flow chromatography (Amersham Pharmacia Biotech))
Yeast culture supernatant (3 L) containing GCSF-HSA encoded by construct 1642 was loaded onto a phenyl fast flow column containing 1 M ammonium sulfate in 50 mM Tris, pH 7.2. The column was washed with 1 M ammonium sulfate in 50 mM Tris (pH 7.2), 0.2 M ammonium sulfate in 50 mM Tris (pH 7.2), and then with buffer. The GCSF-HSA fusion protein was eluted with water (Water For Injected Water WFI).

(Step 2: SP high-speed flow chromatography (Amersham Pharmacia Biotech))
The eluate from step 1 was mixed with an equal volume of solution (composed of 10.3 mM Na ₂ HPO ₄ and 4.85 mM citric acid, pH 5.0). The mixture was loaded onto an SP high-speed flow column and eluted with a solution (consisting of 10.3 mM Na ₂ HPO ₄ and 4.85 mM 0.5 M NaCl in citric acid, pH 5.0). The column was then stripped with a solution (composed of 10.3 mM Na ₂ HPO ₄ and 4.85 mM 1 M NaCl in citric acid, pH 5.0).

(Step 3: Methyl HIC chromatography (BioRad))
The eluate from step 2 was titrated to a final concentration of 1 M ammonium sulfate (143 mS) in 50 mM Tris (pH 7.2) and loaded onto a methyl HIC column. The column was washed to baseline and then with 0.6 M ammonium sulfate in 50 mM Tris (pH 7.2). A gradient from 0.6 M ammonium sulfate to 0 M ammonium sulfate was started. Finally, the column was stripped with WFI and 0.5M NaOH. A large amount of impurities in the sample eluted at a lower ammonium sulfate concentration, thereby giving a high purity GCSF-HSA fusion.

(Step 4: CM high-speed flow chromatography (Amersham Pharmacia Biotech))
The eluate from step 3 was diluted with WFI to 5 mS (pH 5.5) and loaded onto a CM column at 300 cm / hr. The column was eluted with 0.5 mM NaCl (pH 5.5) in 11 mM Na ₂ HPO ₄ and 4 mM citric acid. The column was stripped with 11 mM Na ₂ HPO ₄ and 1 M NaCl (pH 5.5) in 4 mM citric acid.

(Step 5: Ultrafiltration / Diafiltration (Amersham Pharmacia Biotech)
The purified product was ultrafiltered and diafiltered in phosphate buffered saline (“PBS”) (pH 7.2).

  The purified GCSF albumin fusion protein encoded by construct 1642 was analyzed for purity by SDS / PAGE. This was> 95% pure. The protein was sequenced and confirmed and the protein showed 90% purity by N-terminal sequence analysis using the N-terminal sequence of “TPLGP” (SEQ ID NO: 2144).

(The activity of GCSF can be assayed by an in vitro NFS-60 cell proliferation assay)
(Method)
To assess GCSF activity, NSF-60 cells, a myeloid factor-dependent cell line derived from a primary lake Cascitus wild ecotropic virus-induced tumor of NFS mice are used.

(Cell growth and preparation)
Cells were first placed in a T-75 cm ² flask in growth medium (10% fetal bovine serum, “FBS”, 1 × penicillin / streptomycin, 1 × L-glutamine (final concentration 2 mM)) and recombinant mouse interferon. Seed at approximately 1.5 × 10 ⁴ cells / mL in RPMI 1640) containing Leukine-3 (IL3) (30 ng / mL). Cells are split every 2 days anywhere from 1:10 to 1:20 and replated in fresh medium.

(NFS-60 bioassay)
The NFS-60 assay is performed as described in Weinstein et al. (Weinstein et al., 1986, Proc. Natl. Acad. Sci. USA, 83, pp5010-4). Briefly, the day before performing the assay, cells are replated to 1.0 × 10 ^{5 in} fresh assay growth medium containing IL3. The next day, the cells are transferred to a 50 mL conical tube, centrifuged at low speed, and washed twice with plain RPMI without serum or growth factors. The pellet is resuspended in 25 mL followed by cell counting. Cells are centrifuged once more and resuspended in growth medium (above) at working concentration but without IL3. Cells are plated at 1 × 10 ⁵ cells / well in 96 well round bottom TC-treated plates. Increasing doses of GCSF are added to each well to a final volume of 0.1 mL. The assay is performed in triplicate. Cells are cultured for 24 hours and the level of cell proliferation is determined. ³ H-thymidine (5 μCi / mL) is added 4 hours before the end of the experiment. Cells were then onto glass fiber filters, and collected using a cell harvester, and ³ H- the amount of labeled DNA thymidine is counted using a TOP-Count.

(The activity of GCSF albumin fusion encoded by Construct ID # 1642 can be assayed using an in vitro NFS-60 cell proliferation assay)
(Method)
The GSCF albumin fusion protein encoded by construct 1642 was tested in the in vitro NFS-60 cell proliferation bioassay described above.

(Cell growth and preparation)
Cells were prepared as described above.

(NFS-60 bioassay)
The day before performing the assay, cells were replated up to 1.0 × 10 ^{5 in} fresh assay growth medium containing IL3. The next day, the cells were transferred to a 50 mL conical tube, centrifuged at low speed, and washed twice with plain RPMI without serum or growth factors. The pellet was resuspended in 25 mL followed by cell counting. Cells were centrifuged again and resuspended in growth medium (above) at working concentration but without IL3. Cells were plated at 1 × 10 ⁵ cells / well in 96 well round bottom TC treated plates. Increasing doses of either HSA, recombinant human GCSF (rhGCSF) or partially purified GCSF albumin fusion protein from yeast supernatant are added to individual wells to a final volume of 0.1 mL. The assay was performed in triplicate. Cells were cultured for 24 hours and the level of cell proliferation was determined. ³ H-thymidine (5 μCi / mL) was added 4 hours before the end of the experiment. Cells were then collected on a glass fiber filter using a cell collector and ³ H-thymidine labeled DNA was counted using TOP-Count.

(result)
Construct 1642 demonstrated the ability to induce NFS-60 cell proliferation activity in a dose-dependent manner, while control supernatants from yeast expressing HSA alone did not produce any activity (FIG. 11).

(The activity of GCSF can be assayed in vivo using C57BL / 6 mice) (GCSF as fixative)
G-CSF can fix granulocytes around and can increase the total white blood cell (WBC) count when administered to mice. Recombinant human GCSF (rhGCSF) cross-reacts with recombinant mouse GCSF (rmGCSF).

(Method)
Tag the ears of the mice before starting the injection. Mice are injected intraperitoneally with rhGCSF (Neupogen, AMEN) at either 5 g (n = 5) or 10 g (n = 5) twice daily for 7 consecutive days. Control mice (n = 3) are administered Hepes buffered saline (HBSS). Twenty-four hours after the last rhGCSF administration, peripheral blood is collected from the tail and analyzed for granulocyte count and total WBC count.

(result)
Both doses of rhGCSF effectively increase both the frequency and total number of granulocytes, as well as the total WBC count (see FIG. 12). This effect is evident 24 hours after the final intraperitoneal administration of rhGCSF. This effect is transient and the number of granulocytes returns to normal by day 5.

(The activity of the GCSF albumin fusion protein encoded by construct ID # 1642 can be assayed in vivo using C57BL / 6 mice: GCSF-HSA as fixative)
(Method)
The GCSF albumin fusion protein encoded by construct 1642 can be assayed according to the procedure described above. Briefly, the mouse ear is tagged before initiating the injection. Mice are injected intraperitoneally with rhGCSF (as a control) or GCSF albumin fusion protein at either 5 g (n = 5) or 10 g (n = 5) twice daily for 7 consecutive days. Additional control mice (n = 3) receive Hepes buffered saline (“HBSS”). Twenty-four hours after the last GCSF administration, peripheral blood can be collected from the tail and analyzed for granulocyte count and total WBC count.

(Example 20: Construct ID 1643, HSA-GCSF, production)
Construct ID 1643 (pSAC35: HSA.GCSF.T31-P204) contains DNA encoding the GCSF albumin fusion protein, which is the yeast S. cerevisiae. It has a full-length HSA protein containing an HSA chimeric leader sequence (ie, HSA-kex2 signal peptide) fused to the amino terminus of the mature form of GCSF protein (ie, A21-T153) in the cerevisiae expression vector pSAC35.

(GCSF cDNA cloning)
The polynucleotide encoding GCSF was PCR amplified using the primers GCSF-3 and GCSF-4 described below. The amplified product was cleaved with Bsu 36I / Asc I and ligated to pScNHSA cleaved with Bsu 36I / Asc I. Construct ID # 1643 encodes an albumin fusion protein comprising a chimeric leader sequence of HSA, and a mature form of HSA and a mature form of GCSF.

  Two oligonucleotides (GCSF-3 and GCSF-4) suitable for PCR amplification of the polynucleotide encoding the mature form of GCSF were synthesized:

ＧＣＳＦ−３は、Ｂｓｕ ３６Ｉクローニング部位（下線を引いて示される）、およびＨＳＡの成熟形態の最後の４アミノ酸残基をコードするヌクレオチド、ならびにＧＣＳＦの成熟形態の最初の６アミノ酸残基をコードする２０ヌクレオチドを含む。ＧＣＳＦ−４において、Ａｓｃ Ｉ部位は、下線を引いて示され、そして最後の２４ヌクレオチドは、ＧＣＳＦの最後の８アミノ酸残基をコードするＤＮＡの逆相補体である。ＨＳＡ−ＧＣＳＦのＰＣＲ増幅体を、これらのプライマーを使用して産生し、精製し、Ｂｓｕ ３６Ｉ制限酵素およびＡｓｃ Ｉ制限酵素で消化し、そしてｐＳｃＮＨＳＡベクターのＢｓｕ ３６Ｉ部位およびＡｓｃ Ｉ部位にクローニングした。配列を確認した後に、このＧＣＳＦアルブミン融合タンパク質をコードする発現カセットを、Ｎｏｔ ＩフラグメントとしてｐＳＡＣ３５にサブクローニングした。

GCSF-3 encodes the Bsu 36I cloning site (shown underlined), and the nucleotide encoding the last 4 amino acid residues of the mature form of HSA, and the first 6 amino acid residues of the mature form of GCSF Contains 20 nucleotides. In GCSF-4, the Asc I site is underlined and the last 24 nucleotides are the reverse complement of DNA encoding the last 8 amino acid residues of GCSF. PCR amplifications of HSA-GCSF were generated using these primers, purified, digested with Bsu 36I and Asc I restriction enzymes, and cloned into the Bsu 36I and Asc I sites of the pScNHSA vector. After confirming the sequence, the expression cassette encoding this GCSF albumin fusion protein was subcloned into pSAC35 as a Not I fragment.

  Furthermore, analysis by amino acid sequencing of the N-terminus of the expressed albumin fusion protein confirmed the presence of the predicted HSA sequence (see below).

  The GCSF albumin fusion protein of the invention is preferably a mature form of HSA (ie Asp-25) fused to either the N-terminus or C-terminus of the mature form of GCSF (ie Thr-31 to Pro-204). ~ Leu609). In one embodiment of the invention, the GCSF albumin fusion protein of the invention further comprises a signal sequence that directs the nascent fusion polypeptide in the secretory pathway of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed and the mature GCSF albumin fusion protein is secreted directly into the culture medium. The GCSF albumin fusion protein of the present invention includes heterologous signal sequences (MAF, INV, Ig, Fibulin B, Clusterin, insulin-like growth factor binding protein 4, modified HSA leader sequences (including chimeric HSA / MAF leader sequences, Or other heterologous signal sequences known in the art, including but not limited to). In a preferred embodiment, the GCSF albumin fusion protein of the invention comprises a native GCSF signal sequence. In a further preferred embodiment, the GCSF albumin fusion protein of the present invention further comprises an N-terminal methionine residue. Polynucleotides (including fragments and / or variants) encoding these polypeptides are also encompassed by the present invention.

(Expression and purification of construct ID1643)
(Expression in yeast S. cerevisiae)
For construct 1643, S.M. Transformation into cerevisiae was performed by methods known in the art (see Example 3) and as previously described for construct ID 1642 (see Example 19).

(Purification from the supernatant of yeast S. cerevisiae cells)
The general procedure for the purification of albumin fusion protein is described in Example 4. Yeast S. The cell supernatant containing the GCSF albumin fusion protein expressed from construct ID no. 1643 in cerevisiae cells was purified according to the following method. Another purification scheme is described in Example 19.

(Step 1: Phenyl Sepharose rapid flow (hs), pH 7.2)
The fermentation supernatant (3.5 L) was adjusted to 139 mS and pH 7.2 with ammonium sulfate to a final concentration of 1 M in 50 mM Tris (pH 7.2). A phenyl sepharose column was packed at a flow rate of 300 cm / hr. The column was washed with 50 mM Tris-HCl (pH 7.2). A series of low salt elutions were performed to remove contaminating proteins, followed by elution of the target protein by WFI elution. NaOH removal of this column revealed that a significant portion of the target protein was not removed by the previous treatment.

(Step 2: Mimic Blue, pH 6.5)
The eluted target protein is diafiltered with 20 mM citrate phosphate buffer (CPB) (pH 6.5) and then applied to a Mimic Blue column pre-equilibrated with 20 mM CPB (pH 6.5) buffer. Filled. The column was washed with equilibration buffer at 10 column volumes. The majority of the target protein was then eluted with a 0.2M NaCl wash. Higher salt concentration elution solutions (1M and 2M NaCl) revealed some target protein. However, when HPLC-SEC was performed on these fractions, most of the target protein was observed as aggregates. This purification step yielded a target protein that was more than 85% pure.

(Step 3: Q HP, pH 6.5)
The target protein was diluted with 20 mM CPB (pH 6.5) (5-fold) to a conductivity of less than 5 mS and loaded onto Q HP resin. A series of elutions (100 mM, 200 mM, 500 mM, and 1 M NaCl) were performed. The target protein was eluted with 100 mM NaCl.

(Step 4: SP FF, pH 5.5)
The target protein was diluted with 20 mM CPB (pH 5.0) and the pH was adjusted to 5.0. The target protein was loaded onto an SP Sepharose FF column. The column was washed with 5 column volumes of equilibration buffer. A 45 kDa contaminating protein (proteolytic fragment of HSA) did not bind to the resin and was observed in the packed flow through (LFT). The target protein eluted with a shallow slope of 0-500 mM NaCl. The target protein was eluted with approximately 250 mM NaCl. The target protein was diafiltered into a final storage buffer of 20 mM CPB (pH 6.5).

  Analysis by SDS-PAGE identified an 88 kDa protein more than 95% pure. As a result of N-terminal sequencing, the major sequence is “DAHKS” (SEQ ID NO: 2143), which is the amino terminus of the mature form of HSA. The final buffer composition is 20 mM CPB (pH 6.5). From 3.5 L of culture supernatant, 1.94 mg of protein was purified.

(The activity of the GCSF albumin fusion protein encoded by construct ID number 1643 can be assayed using an in vitro NFS-60 cell proliferation assay.)
(Method)
GCSF albumin fusion protein encoded by construct 1643 is sub-titled “GCSF activity can be assayed using in vitro NFS-60 cell proliferation assay” and “GCSF albumin fusion encoded by construct ID number 1642. The activity of can be assayed using an in vitro NFS-60 cell proliferation assay and was tested in the in vitro NFS-60 cell proliferation bioassay previously described in Example 19.

(result)
Construct 1643 demonstrated the ability to cause NFS-60 cell proliferation in a dose-dependent manner, while control supernatants using HSA alone did not produce any activity (see FIG. 11). about).

(The activity of the GCSF albumin fusion encoded by construct ID number 1643 can be assayed in vivo using C57BL / 6 mice with GCSF-HSA as a modifying agent.)
(Method)
The GCSF albumin fusion protein encoded by construct 1643 is a subsection titled “The activity of GCSF can be assayed in vivo using C57BL / 6 mice with GCSF-HSA as a modifying agent” and “construct ID number. The activity of the GCSF albumin fusion encoded by 1642 can be assayed in vivo using C57BL / 6 mice with GCSF-HSA as a modifying agent. "The procedure described above in Example 19 below. Can be assayed according to

(Example 21: Indication of GCSF albumin fusion protein)
Based on the activity of the GCSF albumin fusion protein in the above assay, the GCSF albumin fusion protein can be used for inflammatory disorders, osteoblast leukemia, primary neutropenia (eg, Kostmann syndrome), secondary neutropenia, favorable Prevention of neutropenia, prevention and treatment of neutropenia in HIV-infected patients, prevention and treatment of neutropenia associated with chemotherapy, infection associated with neutropenia, myelopysplasia ), And chemoprevention of autoimmune disorders, hematopoietic progenitor cell mobilization, bone marrow transplantation, acute myeloid leukemia, non-Hodgkin lymphoma, acute lymphoblastic leukemia, Hodgkin's disease, enhanced bone marrow recovery, and glycogen storage disease, Useful in treatment, prevention, and / or diagnosis.

(Example 22: Construct ID 2363, GFSF-HSA-EPO.A28-D192, production)
Construct ID 2363 (pC4: GCSF.HSA.EPO-A28-D192) contains DNA encoding the GCSF-HSA-EPO triple fusion protein. This triple fusion protein is a protein having the full length form of granulocyte colony stimulating factor (G-CSF) and fused to the amino terminus of the mature form of HSA, which is the amino terminus of the mature form of EPO (ie , Amino acids A28-D192), with the exception of the last Arg residue in the CHO mammalian cell line expression vector pC4.

(Cloning of GCSF-HSA-EPO cDNA)
Construct ID number 1642, ie pSAC35: HGCSF. T31-P204. A portion of construct 2363 was produced using HSA (Example 19) as a template. The following polynucleotides were synthesized:

ＧＣＳＦの全長配列を、プライマーＧＣＳＦ／ＥＰＯ−１、ＧＣＳＦ／ＥＰＯ−２、ＧＣＳＦ／ＥＰＯ−３、およびＧＣＳＦ／ＥＰＯ−４の組み合わせを使用して、３段階オーバーラッピングＰＣＲ反応において産生した。プライマーＧＣＳＦ／ＥＰＯ−１、ＧＣＳＦ／ＥＰＯ−３およびＧＣＳＦ／ＥＰＯ−４は、ＧＣＳＦの全長のアミノ末端にまたがる配列からなる。プライマーＧＣＳＦ／ＥＰＯ−２は、ＨＳＡのアミノ酸Ｓｅｒ−２１６〜Ａｌａ−２２５にまたがる配列の逆相補体からなる。第一のＰＣＲ反応は、構築物１６４２をテンプレートとして、ならびにプライマーＧＣＳＦ／ＥＰＯ−１およびＧＣＳＦ／ＥＰＯ−２を含んだ。第一のＰＣＲ反応から得られた産物を、第二のＰＣＲ反応においてテンプレートとして使用し、この反応は、プライマーＧＣＳＦ／ＥＰＯ−３およびＧＣＳＦ／ＥＰＯ−２を含んだ。第二のＰＣＲ反応から得られた産物を、第三のＰＣＲ反応においてテンプレートとして使用し、この反応は、プライマーＧＣＳＦ／ＥＰＯ−４およびＧＣＳＦ／ＥＰＯ−２を含んだ。プライマーＧＣＳＦ／ＥＰＯ−４は、Ｂａｍ ＨＩ部位（斜体で示される）、引き続いてＫｏｚａｋ配列（下線を引かれて示される）を有する。最後のＰＣＲ産物は、５’Ｂａｍ ＨＩ制限部位、引き続いて適切なＫｏｚａｋ配列、その全長ＧＣＳＦコード配列全体およびＨＳＡオープンリーディングフレーム（Ａｓｐ−２５〜Ａｌａ−２２５）の一部を含む。Ｃｌａ Ｉ部位は、ＨＳＡの成熟形態のポリヌクレオチド配列に固有であり、そしてＨＳＡの成熟形態の５’末端のすぐ近くに位置する。Ｂａｍ ＨＩ−Ｃｌａ Ｉフラグメントを、同様に消化したｐＣ４．ＨＳＡ．ＥＰＯ．Ａ２８−Ｄ１９２構築物ＩＤ番号１９８１にクローニングした。

The full length sequence of GCSF was generated in a three-step overlapping PCR reaction using a combination of primers GCSF / EPO-1, GCSF / EPO-2, GCSF / EPO-3, and GCSF / EPO-4. Primers GCSF / EPO-1, GCSF / EPO-3 and GCSF / EPO-4 consist of sequences spanning the amino terminus of the full length of GCSF. Primer GCSF / EPO-2 consists of the reverse complement of the sequence spanning amino acids Ser-216 to Ala-225 of HSA. The first PCR reaction included construct 1642 as a template and primers GCSF / EPO-1 and GCSF / EPO-2. The product obtained from the first PCR reaction was used as a template in the second PCR reaction, which included primers GCSF / EPO-3 and GCSF / EPO-2. The product obtained from the second PCR reaction was used as a template in the third PCR reaction, which included primers GCSF / EPO-4 and GCSF / EPO-2. Primer GCSF / EPO-4 has a BamHI site (shown in italics) followed by a Kozak sequence (shown underlined). The final PCR product contains a 5 ′ Bam HI restriction site followed by the appropriate Kozak sequence, its entire full-length GCSF coding sequence and a portion of the HSA open reading frame (Asp-25-Ala-225). The Cla I site is unique to the polynucleotide sequence of the mature form of HSA and is located immediately adjacent to the 5 ′ end of the mature form of HSA. The Bam HI-Cla I fragment was similarly digested with pC4. HSA. EPO. It was cloned into A28-D192 construct ID number 1981.

  Construct ID number 2363 encodes an albumin fusion protein comprising the leader and mature form of GCSF, followed by the mature HSA protein, followed by the mature form of EPO.

  Furthermore, analysis of the N-terminus of the expressed albumin fusion protein by amino acid sequencing can confirm the presence of the predicted GCSF sequence (see below).

  The GCSF / EPO albumin fusion protein of the invention is preferably fused to either the N-terminus or C-terminus of the mature form of GCSF (ie, Thr-31 to Pro-204), and the mature form of EPO (ie, A mature form of HSA (ie Asp-25 to Leu-609) fused to either the N-terminus or C-terminus of Ala-28 to Asp-192). In one embodiment of the invention, the GSCSF / EPO albumin fusion protein of the invention further comprises a signal sequence that directs the nascent fusion polypeptide in the host secretory pathway used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed and the mature GCSF / EPO albumin fusion protein is secreted directly into the culture medium. The GCSF / EPO albumin fusion protein of the present invention has a heterologous signal sequence (MAF, INV, Ig, Fibulin B, Clusterin, insulin-like growth factor binding protein 4, modified HSA leader sequence (including chimeric HSA / MAF leader sequence). , Or other heterologous signal sequences known in the art, but are not limited thereto). In a preferred embodiment, the GCSF / EPO albumin fusion protein of the invention comprises either a native GCSF signal sequence or a native EPO signal sequence. In a further preferred embodiment, the GCSF / EPO albumin fusion protein of the invention further comprises an N-terminal methionine residue. Polynucleotides (including fragments and / or variants) encoding these polypeptides are also encompassed by the present invention.

(Expression and purification of construct ID 2363)
(Expression in CHO cells)
Construct 2363 can be transfected into CHO cells as previously described in Examples 6 and 8.

(Purification from CHO supernatant)
The general purification procedure for albumin fusion protein is described in Example 7. The triple fusion protein GCSF-HSA-EPO encoded by construct 2363 can be purified as described above in Examples 7 and 9. N-terminal sequencing should yield the sequence TPLGP (SEQ ID NO: 2144), corresponding to the mature form of GCSF.

(The activity of GCSF-HSA-EPO encoded by Construct ID # 2363 can be assayed using in vitro TF-1 cell proliferation assay and in vitro NFS-60 cell proliferation assay.)
(Method)
The activity of the triple fusion protein GCSF-HSA-EPO encoded by construct 2363 was measured in vitro as described above in Example 9 under the subsection title “In Vitro TF-1 Cell Proliferation Assay for Construct 1981”. In the TF-1 cell proliferation assay and in Example 19, the subsection title “The activity of the GCSF albumin fusion encoded by construct ID number 1642 can be assayed using an in vitro NFS-60 cell proliferation assay”. Assayed in an in vitro NFS-60 cell proliferation assay as previously described below.

(result)
The GCSF-HSA-EPO albumin fusion encoded by construct 2363 demonstrated the growth of both TF-1 and NFS-60 cells.

(The activity of the GCSF-HSA-EPO albumin fusion encoded by Construct ID number 2363 can be assayed in vivo.)
The activity of the triple fusion protein GCSF-HSA-EPO encoded by construct 2363 can be assayed using the in vivo Harlan mouse model for measuring hematocrit in the subsection heading “Activity of construct 1981” in Example 9. In the in vivo Harlan mouse model and in C57BL / 6 mice (where GCSF-HSA-EPO is a mobilizing agent) as described previously under The activity of the GCSF albumin fusion encoded by ID No. 1642 was assayed as described previously under “Using C57BL / 6 mice: GCSF-HSA can be assayed in vivo”. Hematocrit levels can be measured.

(Example 23: Construct ID 2373, GCSF-HSA-EPO.A28-D192, production)
Construct ID 2373 (pC4: GCSF.HSA.EPO-A28-D192.R140G) contains DNA encoding the GCSF-HSA-EPO triple fusion protein. This triple fusion protein is a protein that has the full length form of granulocyte colony stimulating factor “G-CSF” and is fused to the amino terminus of the mature form of HSA, which is the amino terminus of the mature form of EPO (ie , A28-D192), and has a mutation of Arg-140 to Gly in the CHO mammalian cell line expression vector pC4.

(Cloning of EPO cDNA for construct 2373)
Construct ID number 2373 encodes an albumin fusion protein containing the leader sequence and mature form of GCSF, followed by the mature HSA protein, followed by the mature form of EPO, which has a mutation from Arg-140 to Gly. (SEQ ID NO: 401). Construct ID number 2373 was generated using construct ID number 2363 (ie, pC4: GCSF.HSA.EPO.R140G) as a template for PCR mutagenesis.

  Four oligonucleotides (GCSF / EPO-5, GCSF / EPO-6, GCSF / EPO-7, and GCSF / EPO-8) suitable for PCR amplification of the template with construct ID number 2363 were synthesized.

構築物２３７３を、ネスティッド（ｎｅｓｔｅｄ）ＰＣＲ増幅によって、構築物２３６３をテンプレートとして使用して産生した。第一回のＰＣＲ増幅において、構築物ＩＤ２３６３のＮ末端フラグメントおよびＣ末端フラグメントを、独立して増幅した。Ｎ末端フラグメントを、プライマーＧＣＳＦ／ＥＰＯ−５およびＧＣＳＦ／ＥＰＯ−６を使用して産生した。ＧＣＳＦ／ＥＰＯ−５は、ＨＳＡの全長形態のアミノ酸残基３３４〜３４０をコードする核酸配列に対応する。ＧＣＳＦ／ＥＰＯ−６プライマーは、ＥＰＯの全長形態のアミノ酸１３６〜１４３にわたる配列の逆相補体を含む（アミノ酸１４０におけるＡｒｇ残基をコードするコドンＣＧＡ（太字で強調されている）がＧｌｙ残基をコードするコドンＧＧＡに変更されていることを例外とする）。Ｃ末端フラグメントを、プライマーＧＣＳＦ／ＥＰＯ−７およびＧＣＳＦ／ＥＰＯ−８を使用して産生した。ＧＣＳＦ／ＥＰＯ−７プライマーは、ＥＰＯの全長形態のアミノ酸１３６〜１４３をコードする核酸配列を含む（アミノ酸１４０におけるＡｒｇ残基をコードするコドンＣＧＡ（太字で強調されている）がＧｌｙ残基をコードするコドンＧＧＡに変更されていることを例外とする）。ＧＣＳＦ／ＥＰＯ−８において、この配列は、終止コドンの下流のｐＣ４ベクター内のヌクレオチドを含む。第二回のＰＣＲ増幅において、プライマーＧＣＳＦ／ＥＰＯ−５およびＧＣＳＦ／ＥＰＯ−８を使用して、ＧＣＳＦ−ＨＳＡ−ＥＰＯ三重融合タンパク質を増幅した。この三重融合タンパク質は、ＨＳＡの成熟形態のアミノ末端に融合するＧ−ＣＳＦの全長形態を有し、このＨＳＡは、Ａｒｇ−１４０からＧｌｙへの変異を有するＥＰＯの全長形態のアミノ末端（すなわち、Ａ２８−Ｄ１９２）に融合している。この反応混合物は、ＰＣＲ増幅されたＮ末端フラグメントおよびＰＣＲ増幅されたＣ末端フラグメントの両方を含んだ。

Construct 2373 was produced by nested PCR amplification using construct 2363 as a template. In the first round of PCR amplification, the N- and C-terminal fragments of construct ID 2363 were amplified independently. N-terminal fragments were generated using primers GCSF / EPO-5 and GCSF / EPO-6. GCSF / EPO-5 corresponds to the nucleic acid sequence encoding amino acid residues 334-340 of the full-length form of HSA. The GCSF / EPO-6 primer contains a reverse complement of the sequence spanning amino acids 136-143 of the full-length form of EPO (codon CGA encoding the Arg residue at amino acid 140 (highlighted in bold)) but with a Gly residue. The exception is that the coding codon is changed to GGA). C-terminal fragments were generated using primers GCSF / EPO-7 and GCSF / EPO-8. The GCSF / EPO-7 primer contains a nucleic acid sequence encoding amino acids 136-143 of the full-length form of EPO (codon CGA encoding the Arg residue at amino acid 140 (highlighted in bold) encodes a Gly residue) Except that it is changed to codon GGA). In GCSF / EPO-8, this sequence includes nucleotides in the pC4 vector downstream of the stop codon. In the second round of PCR amplification, primers GCSF / EPO-5 and GCSF / EPO-8 were used to amplify the GCSF-HSA-EPO triple fusion protein. This triple fusion protein has the full-length form of G-CSF fused to the amino terminus of the mature form of HSA, which is the amino terminus of the full-length form of EPO with the Arg-140 to Gly mutation (ie, A28-D192). The reaction mixture contained both a PCR amplified N-terminal fragment and a PCR amplified C-terminal fragment.

  The PCR product was purified and then digested with Bsu36I and AscI. After further purification of the Bsu36I-AscI fragment by gel electrophoresis, this product was cloned into Bsu36I / AscI digested construct 2363 to yield construct ID number 2373.

  In addition, analysis of the N-terminus of albumin fusion protein by amino acid sequencing can confirm the presence of the predicted GCSF sequence (see below).

  The GCSF / EPO albumin fusion protein of the invention is preferably fused to either the N-terminus or C-terminus of the mature form of GCSF (ie, Thr-31-Pro-240), and the mature form of EPO (ie, A mature form of HSA (ie Asp-25 to Leu-609) fused to either the N-terminus or C-terminus of Ala28-Asp-192). In one embodiment of the invention, the GCSF / EPO albumin fusion protein of the invention further comprises a signal sequence that directs the nascent fusion polypeptide in the secretory pathway of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed and the mature GCSF / EPO albumin fusion protein is secreted directly into the culture medium. The GCSF / EPO albumin fusion protein of the present invention has a heterologous signal sequence (MAF, INV, Ig, Fibulin B, Clusterin, insulin-like growth factor binding protein 4, modified HSA leader sequence (including chimeric HSA / MAF leader sequence). , Or other heterologous signal sequences known in the art, but are not limited thereto). In a preferred embodiment, the GCSF / EPO albumin fusion protein of the invention comprises either a native GCSF signal sequence or a native EPO signal sequence. In a further preferred embodiment, the GCSF / EPO albumin fusion protein of the invention further comprises an N-terminal methionine residue. Polynucleotides (including fragments and / or variants) encoding these polypeptides are also encompassed by the present invention.

(Expression and purification of construct ID 2373)
(Expression in CHO cells)
Construct 2373 can be transfected into CHO cells as previously described in Examples 6 and 8.

(Purification from CHO supernatant)
The general purification procedure for albumin fusion protein is described in Example 7. Triple fusion protein GCSF-HSA-EPO. R140G can be purified as described above in Examples 7 and 8. N-terminal sequencing should yield the sequence TPLGP (SEQ ID NO: 2144), corresponding to the mature form of GCSF.

(The activity of GCSF-HSA-EPO.R140G encoded by Construct ID number 2373 can be assayed using in vitro TF-1 cell proliferation assay and in vitro NFS-60 cell proliferation assay.)
(Method)
Triple fusion protein GCSF-HSA-EPO. The activity of R140G was determined in Example 9 in an in vitro TF-1 cell proliferation assay as described previously under the subsection title “In Vitro TF-1 Cell Proliferation Assay for Construct 1981” and in Example 19. In vitro NFS-60 as described above under the subsection title "The activity of the GCSF albumin fusion encoded by construct ID number 1642 can be assayed using an in vitro NFS-60 cell proliferation assay". It can be assayed in a cell proliferation assay.

(result)
The GCSF-HSA-EPO. The R140G albumin fusion demonstrated the growth of both TF-1 and NFS-60 cells.

(The activity of the GCSF-HSA-EPO.R140G albumin fusion encoded by construct ID number 2373 can be assayed in vivo.)
(Method)
Triple fusion protein GCSF-HSA-EPO. The activity of R140G was determined in vivo as described previously in Example 9 under the subsection title "Activity of construct 1981 can be assayed using an in vivo Harlan mouse model for measuring hematocrit". In the Harlan mouse model, and in C57BL / 6 mice, where GCSF-HSA-EPO is a mobilizing agent, the subsection title “Activity of GCSF Albumin Fusion Encoded by Construct ID No. 1642 in Example 19” Can be assayed and hematocrit levels measured as described previously under "Using C57BL / 6 mice: GCSF-HSA can be assayed in vivo".

Example 24: GCSF-HSA-EPO triple fusion indication
Indications for triple fusion proteins including GCSF, EPO and HSA, including but not limited to those encoded by constructs 2363 and 2373, include indications detailed for EPO albumin fusion proteins and GCSF albumin fusion proteins. Diseases include, but are not limited to, various conditions (end-stage renal disease (dialysis patients), chronic renal failure prior to dialysis, HIV patients treated with zidovudine, cancer patients undergoing chemotherapy, and premature infants. Preoperative in anemia patients undergoing selective non-cardiac nonvascular surgery to reduce the need for blood transfusion; aplastic anemia and other refractory For anemia, refractory anemia in inflammatory bowel disease, and selective orthopedic chemoprotection Transfusion escape; inflammatory disorders, myeloid leukemia, primary neutropenia (eg, Kostmann syndrome), treatment, prevention and / or diagnosis of secondary neutropenia, prevention of neutropenia, Prevention and treatment of neutropenia in HIV-infected patients, prevention and treatment of neutropenia associated with chemotherapy, infection associated with neutropenia, spinal cysts, and autoimmune disorders, hematopoietic progenitor cells Mobilization, bone marrow transplantation, acute myeloid leukemia, non-Hodgkin lymphoma, acute lymphoblastic leukemia, Hodgkin's disease, accelerated bone marrow recovery, and glycogen storage disease.

(Example 25: Construct ID 2053, IFNb-HSA, production)
Construct ID 2053 (pEE12.1: IFNb.HSA) contains DNA encoding an IFNb albumin fusion protein, which fusion protein is fused to the amino terminus of the mature form of HSA in the NS0 expression vector pEE12.1. It has a full length IFNb protein containing sequence.

(Cloning of IFNb cDNA)
A polynucleotide encoding IFNb was PCR amplified using the primers IFNb-1 and IFNb-2 described below, cut with Bam HI / Cla I, and Bam HI / Cla I cut pC4: HSA To give construct 2011. The Eco RI / Eco RI fragment from construct ID number 2011 was subcloned into the Eco RI site of pEE12.1 to produce construct ID number 2053. Construct ID number 2053 contains DNA encoding the albumin fusion protein, including the leader sequence and mature form of IFNb, followed by the mature HSA protein.

  Two oligonucleotides (IFNb-1 and IFNb-2) suitable for PCR amplification of the polynucleotide encoding the full length of IFNb were synthesized:

ＩＦＮｂ−１プライマーは、Ｂａｍ ＨＩクローニング部位（下線を引いて示される）、Ｅｃｏ ＲＩクローニング部位、およびＫｏｚａｋ配列（斜体で示される）、続いてＩＦＮｂの全長形態の最初の２７個のアミノ酸をコードする８０個のヌクレオチドを含む。ＩＦＮｂ−２において、Ｃｌａ Ｉ部位（下線を引いて示される）およびそれに続くＤＮＡは、成熟ＨＳＡタンパク質（配列番号１０３８）の最初の１０個のアミノ酸をコードするＤＮＡの逆相補体であり、そして最後の１８個のヌクレオチドは、ＩＦＮｂの最後の６個のアミノ酸残基をコードするＤＮＡの逆相補体である（実施例２を参照のこと）。ＰＣＲ増幅体（ａｍｐｌｉｍｅｒ）を、３つのプライマーを使用して産生し、精製し、Ｂａｍ ＨＩ制限酵素およびＣｌａ Ｉ制限酵素で消化し、そしてｐＣ４：ＨＳＡベクターのＢａｍ ＨＩ部位およびＣｌａ Ｉ部位にクローニングした。配列を確認した後に、ＩＦＮｂアルブミン融合タンパク質発現カセットを含むＥｃｏ ＲＩフラグメントを、Ｅｃｏ ＲＩ消化されたｐＥＥ１２．１にサブクローニングした。

The IFNb-1 primer encodes the Bam HI cloning site (shown underlined), the Eco RI cloning site, and the Kozak sequence (shown in italics) followed by the first 27 amino acids of the full-length form of IFNb Contains 80 nucleotides. In IFNb-2, the Cla I site (shown underlined) and subsequent DNA is the reverse complement of DNA encoding the first 10 amino acids of the mature HSA protein (SEQ ID NO: 1038), and finally Of 18 nucleotides is the reverse complement of DNA encoding the last 6 amino acid residues of IFNb (see Example 2). A PCR amplifier was generated using three primers, purified, digested with Bam HI and Cla I restriction enzymes, and cloned into the Bam HI and Cla I sites of the pC4: HSA vector. . After confirming the sequence, the Eco RI fragment containing the IFNb albumin fusion protein expression cassette was subcloned into Eco RI digested pEE12.1.

  Furthermore, analysis by amino acid sequencing of the N-terminus of the expressed albumin fusion protein can confirm the presence of the predicted IFNb sequence (see below).

  The IFNb albumin fusion protein of the invention is preferably a mature form of HSA (ie Asp-25) fused to either the N-terminus or C-terminus of the mature form of IFNb (ie Met-22 to Asn-187). ~ Leu-609). In one embodiment of the invention, the IFNb albumin fusion protein of the invention further comprises a signal sequence that directs the nascent fusion polypeptide in the secretory pathway of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed and the mature IFNb albumin fusion protein is secreted directly into the culture medium. The IFNb albumin fusion protein of the present invention includes heterologous signal sequences (MAF, INV, Ig, Fibulin B, Clusterin, insulin-like growth factor binding protein 4, modified HSA leader sequences (including chimeric HSA / MAF leader sequences, Or other heterologous signal sequences known in the art, including but not limited to). In a preferred embodiment, the IFNb albumin fusion protein of the invention comprises native IFNb. In a further preferred embodiment, the IFNb albumin fusion protein of the invention further comprises an N-terminal methionine residue. Polynucleotides (including fragments and / or variants) encoding these polypeptides are also encompassed by the present invention.

(Expression and purification of construct ID 2053)
(Expression in mouse myeloma NS0 cell line)
Construct ID number 2053 (pEE12.1: IFNb-HSA) was electroporated into NS0 cells by methods known in the art (see Example 6).

(Purification from NS0 cell supernatant)
Purification of IFNb-HSA from NS0 cell supernatant can be according to the method described in Example 10. This method involves Q-Sepharose anion exchange chromatography at pH 7.4 using a 0-1 M NaCl gradient in 20 mM Tris-HCl followed by a Poros at pH 6.5 using a 5-40 mM sodium citrate gradient. Includes PI 50 anion exchange chromatography and diafiltration for 6 DV to a final buffer concentration of 10 mM citrate (pH 6.5) and 140 mM NaCl. N-terminal sequencing should yield the sequence MSYNLL, which is the amino terminus of the mature form of IFNb. This protein has an approximate MW of 88.5 kDa.

For larger scale purification (eg, 50 L NS0 cell supernatant), it can be concentrated to about 8-10 L. The concentrated sample was then passed through a Q-Sepharose anion exchange column (10 × 19 cm, 1.5 L) using a step elution consisting of 50 mM NaOAc (pH 6.0) and 150 mM NaCl at pH 7.5. Can be done. The eluted sample can then be virus inactivated with 0.75% Triton-X 100 for 60 minutes at room temperature. SDR-reverse phase chromatography (10 cm × 10 cm, 0.8 L) is then used at pH 6.0 with 50 mM NaOAc and 150 mM NaCl, or vice versa, and the sample is loaded onto an SP-Sepharose column at pH 4.8. Can be passed using a step elution of 50 mM NaOAc (pH 6.0) and 150 mM NaCl. Subsequently, DV50 is filtered to remove all virus components. Subsequently, Phenyl-650M chromatography (20 cm × 12 cm, 3.8 L) was used with step elution consisting of 350 mM (NH ₄ ) ₂ SO ₄ and 50 mM NaOAc, or step elution consisting of 50 mM NaOAc (pH 6.0). At pH 6.0. Diafiltration for 6-8 DV is achieved by buffering the buffer at the desired final concentration (10 mM Na ₂ HPO ₄ +58 mM sucrose + 120 mM NaCl (pH 7.2) or 10 mM citric acid (pH 6.5) and 140 mM NaCl or Exchange to 25 mM Na ₂ HPO ₄ , 100 mM NaCl (pH 7.2)).

(The activity of IFNb can be assayed using an in vitro ISRE-SEAP assay.)
All type I interferon proteins are responsive to an interferon (“IFN”) via a common receptor complex and a similar Jak / STAT signaling pathway, which is ultimately via an interferon sequence responsive element (“ISRE”). Signal transduction via gene activation). A convenient assay for type I IFN activity is a promoter-reporter based assay system that contains multiple copies of the ISRE element fused to a downstream reporter gene. A stable HEK293 cell line can be produced and contains a stably integrated copy of the ISRE-SEAP reporter gene, which is very sensitive to type I IFN and is a linear over a 5 log concentration. Showing gender.

(Method of screening for IFNb-HSA NS0 stable clone)
Construct 2053 was electroporated into NS0 cells as described in Example 6. NS0 cells transfected with construct ID no. 2053 were screened for activity by testing growth medium conditioned in ISRE-SEAP assay. ISRE-SEAP / 293F reporter cells were plated at 3 × 10 ⁴ cells / well in 96-well poly D-lysine coated plates one day prior to treatment. Reporter cells are treated with IFNb albumin fusion protein encoded by Construct ID 2053 or various dilutions of rhIFNb conditioned supernatant or purified preparations as controls, including but not limited to 1: 500 and 1: 5000. Processed. The reporter cells were then incubated for 24 hours, after which 40 L was removed for use in the SEAP reporter gene chemiluminescence assay (Roche catalog number 1779842). Recombinant human interferon β (“rhIFNb” (Biogen)) was used as a positive control.

(result)
A purified preparation of NS0-expressed IFNb-HSA has a larger EC50 (9.3 × 10 ⁻⁹ g / mL) than rhIFNb (Biogen), which had an EC50 of 1.8 × 10 ⁻¹⁰ g / mL. ) (See FIG. 13).

(In vivo introduction of OAS by interferon)
(Method)
The OAS enzyme (2′-5′-oligoadenylate synthetase) is activated at the transcriptional level by interferon in response to antiviral infection. The effects of interferon constructs can be measured by obtaining blood samples from treated monkeys and analyzing these samples for transcriptional activation of two OAS mRNAs (p41 and p69). A 0.5 mL volume of leukocytes from 4 animals per group from 7 different time points (0 day, 1 day, 2 days, 4 days, 8 days, 10 days per animal) Day, and day 14). Various groups received vehicle control injections intravenously and / or subcutaneously on day 1, 30 g / kg and / or 300 g / kg of IFN albumin fusion protein, and on days 1, 3, and 5 On the day, it may include subcutaneous injection of 40 g / kg interferon alpha (Schering-Plough) as a positive control. The levels of p41 and p69 mRNA transcripts can be determined by real-time quantitative PCR (Taqman) using probes specific for p41-OAS and p69-OAS. OAS mRNA levels can be quantified relative to 18S ribosomal RNA endogenous control.

(In vivo introduction of OAS by interferon beta albumin fusion encoded by construct ID 2053)
(Method)
The activity of the HSA-IFNb fusion protein encoded by construct 2053 can be assayed in an in vivo OAS assay as described above under the subsection title “In Vivo Introduction of OS by Interferon”.

(Example 26: Indication for IFNb albumin fusion protein)
IFN beta albumin fusion proteins (including but not limited to those encoded by construct 2053) can be used to treat, prevent, reduce and / or detect multiple sclerosis. Other indications include melanoma, solid tumors, cancer, bacterial infection, chemoprotection, embolism, HIV infection, prostate cancer, cancer, hematological malignancies, hematological disorders, preleukemia, glia Tumor, hepatitis B, hepatitis C, human papillomavirus, pulmonary fibrosis, age-related macular degeneration, brain cancer, glioma polymorphism, liver cancer, malignant melanoma, colorectal cancer, Crohn's disease, neurodegeneration, non-small Examples include, but are not limited to, cell lung cancer, rheumatoid arthritis, and ulcerative colitis.

(Example 27: Construct ID 1941, HSA-PTH84, production)
Construct ID 1941 (pC4.HSA.PTH84) encodes an HSA-PTH84 fusion protein, which is the mature form of Ser-1 of human parathyroid hormone ("PTH84") cloned into the mammalian expression vector pC4. Contains the full length of HSA including the native HSA leader sequence fused to ~ Gln-84.

(Cloning of PTH84 cDNA for construct 1941)
DNA encoding PTH84 is amplified using primers PTH84-1 and PTH84-2 described below, cut with Bsu 36I / Not I, and ligated to Bus 36I / Not I cut pC4: HSA did. Construct ID number 1941 encodes an albumin fusion protein comprising the full-length form of HSA, including the native HSA leader sequence followed by the mature PTH84 protein.

  Two appropriate primers (PTH84-1 and PTH84-2) were synthesized for PCR amplification of the polynucleotide encoding the mature form of PTH84.

ＰＴＨ８４−１は、Ｂｕｓ−３６Ｉクローニング部位（下線を引いて示される）、続いてＰＴＨ８４の成熟形態のアミノ酸残基Ｓｅｒ−１〜Ａｓｎ−１０と結合体化した、ＨＳＡの成熟形態の末端（最後のＬｅｕが存在しない）に対応するアミノ酸残基Ａｌａ−Ｌｅｕ−Ｇｌｙをコードする核酸配列を含む。ＰＴＨ８４−２において、Ｎｏｔ Ｉ部位は、下線を引いて示され、そしてそれに続く核酸配列は、成熟ＰＴＨ８４タンパク質の最後の１１個のアミノ酸をコードするＤＮＡの逆相補体に対応する。これらの２つのプライマーを使用して、ＰＴＨ８４タンパク質をＰＣＲ増幅した。

PTH84-1 is the end of the mature form of HSA conjugated to the Bus-36I cloning site (shown underlined) followed by amino acid residues Ser-1 to Asn-10 of the mature form of PTH84 (last The nucleic acid sequence encoding the amino acid residue Ala-Leu-Gly corresponding to In PTH84-2, the Not I site is underlined and the subsequent nucleic acid sequence corresponds to the reverse complement of DNA encoding the last 11 amino acids of the mature PTH84 protein. Using these two primers, the PTH84 protein was PCR amplified.

  Furthermore, analysis by amino acid sequencing of the N-terminus of the expressed albumin fusion protein confirmed the presence of the predicted HSA sequence (see below).

  The PTH84 albumin fusion protein of the invention is preferably a mature form of HSA (ie Asp-25) fused to either the N-terminus or C-terminus of the mature form of PTH84 (ie Ser-1 to Gln-84). ~ Leu-609). In one embodiment of the invention, the PTH84 albumin fusion protein of the invention further comprises a signal sequence that directs the nascent fusion polypeptide in the secretory pathway of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed and the mature PTH84 albumin fusion protein is secreted directly into the culture medium. The PTH84 albumin fusion protein of the present invention includes heterologous signal sequences (MAF, INV, Ig, Fibulin B, Clusterin, insulin-like growth factor binding protein 4, modified HSA leader sequences (including chimeric HSA / MAF leader sequences, Or other heterologous signal sequences known in the art, including but not limited to). In a preferred embodiment, the PTH84 albumin fusion protein of the invention comprises native PTH84. In a further preferred embodiment, the PTH84 albumin fusion protein of the present invention further comprises an N-terminal methionine residue. Polynucleotides (including fragments and / or variants) encoding these polypeptides are also encompassed by the present invention.

(Expression and purification of construct ID 1941)
(Expression in 293T cells)
Construct 1941 was transfected into 293T cells by methods known in the art (eg, Lipofectamine transfection) and selected with 100 nM methotrexate (see Example 6). Expression levels were tested by immunoblot detection using anti-HSA serum as the primary antibody.

(Purification from 293T cell supernatant)
The 293T cell supernatant containing secreted HSA-PTH84 fusion protein expressed in construct ID numbers 1941 to 293T cells was purified as described in Example 7. Specifically, initial capture was performed using anionic HQ-50 resin at pH 7.2, sodium phosphate buffer (20 mM Na ₂ HPO ₄ (pH 7.2)) and 0-0.5 M NaCl. Hydrophobic interaction chromatography using 16 column volumes of salt gradient elution followed by Phenyl 650M resin (Tosohaas) using salt gradient elution of 36 column volumes of 2.75-0M NaCl, pH 7.2. ("HIC"). Here, the sample had a final conductivity of 180 mS. The sample was concentrated using HQ Poros 50 resin and 0.15M NaCl incremental salt step elution. The final buffer composition consisted of 25 mM Na ₂ HPO ₄ +150 mM NaCl (pH 7.2). N-terminal sequencing yielded the amino terminal sequence of the mature form of HSA (ie, DAHKS, SEQ ID NO: 2143). An approximate MW protein of 78 kDa was obtained. A final yield of 0.78 mg protein per liter of 293T cell supernatant was obtained.

(In vitro introduction of cyclic AMP in SaOS2 cells)
(Method)
The biological activity of PTH84 albumin fusion protein can be measured in an in vitro assay in which SaOS-2 (osteosarcoma cell line) is used. PTH activates adenylate cyclase, thereby increasing intracellular cyclic AMP levels.

(Introduction of cAMP in SaOS-2 cells :)
SaOS-2 cells are subcultured at a density of 8.0 × 10 ⁴ cells / well 24 hours before the start of the experiment. On the day of the experiment, cells are serum starved for 2 hours and then treated with a positive control (eg, 5 mg / mL forskolin), recombinant PTH, or PTH albumin fusion protein for 10 minutes. After treatment, these cells are rinsed and intracellular cyclic AMP is extracted with cold ethanol. The ethanol extract is lyophilized and stored at −80 ° C. for further use. The amount of cyclic AMP present in the sample is quantified by ELISA according to the manufacturer's protocol (Amersham Life Sciences, Inc.).

(In vitro introduction of cyclic AMP in SaOS2 cells by albumin fusion protein encoded by construct 1941)
(Method)
An in vitro assay for measuring the introduction of cyclic AMP in SaOS2 cells by the PTH albumin fusion protein encoded by construct 1941 can be performed as described above.

(In vivo: Calcium-induced release in TPTX animals)
(Method)
By monitoring PTH activity, the ability of PTH albumin fusion protein to reduce bone mineral deprivation after thyroid parathyroidectomy ("TPTX") on a low calcium diet and parathyroid hormone treatment, test.

  These animals are described in Votta et al., 1997, J. MoI. Bone and Mineral Res. , 12: 1396-1406; Millest et al., 1997, Bone, 20: 465-471; and Iwata et al., 1997, Arthritis and Rheumatism, 40: 499-509, variability in pharmacological responses. Show. Therefore, between 5 and 8 thyroid parathyroidectomy animals (purchased from an external vendor) per group are used. These animals receive supplemental injections of thyroxine every other day. Each experiment includes several groups: (1) placebo and thyroid hormone (PTH 1-34) injection groups (corresponding to negative and positive controls, respectively); (2-5) PTH albumin fusion protein, Injected intravenously, intraperitoneally, subcutaneously and intramuscularly at various concentrations ranging from 0.1-12 μg / kg before, during or after parathyroid hormone treatment; (6) for some experiments Cysteine protease inhibitors are tested.

  Under isofluorane anesthesia, the left femoral vein and either the left femoral artery or thyroid artery are cannulated with PE-10 tubing fused to heparinized saline PE-50 polyethylene tubing. These catheters are passed subcutaneously, out at the neck and secured to the skin. The animals are allowed to recover for about 18 hours before being used for the experiment, during which time they are fed a calcium-free diet. During the course of the experiment, three blood samples (200 mL each) are taken via thyroid or femoral catheters at 2, 4, and 6 hours after infusion. Longer time points (eg, 18 hours) may also be desirable.

  A dose relationship was established between the appearance of human PTH 1-34, positive control, and ionized calcium levels in whole blood (data not shown).

(The activity of the albumin fusion protein encoded by construct 1941 can be assayed using TPTX animals.)
The activity of the PTH albumin fusion protein encoded by construct 1941 can be measured using the TPTX animals, and the in vivo assay described above under the heading “In Vivo: Induced Release of Calcium in TPTX Animals” .

(In vivo ovariectomized female rat model)
(Method)
PTH activity is tested by monitoring the ability to induce bone formation in ovariectomized female Lewis rats or Sprague Dawley rats.

  Surgery is performed on 8-9 week old female Lewis or Sprague Dawley rats and experiments are not started until 7-10 days after surgery. Samples from blood, urine and left tibia are obtained weekly from 9-12 animals per group. The various groups were sham controls injected with saline daily for 4 weeks, ovariectomized rats injected with saline daily for 4 weeks, and rat PTH peptides 1-34 injected subcutaneously at 10 g / kg 5 times weekly. Ovariectomized rats may be included. Following the fourth and final week of tissue collection, the tibia is sent to Sleetech for bone densitometry analysis.

  Parameters tested were body weight, bone density measurement in the left tibia in 70% ethanol, serum pyridinoline from blood, and urinary deoxypyridinoline and alpha helical protein. Urine samples are taken in the morning. Blood is obtained by bleeding the heart and serum is saved for ELISA analysis. Bone density measurements are performed on the proximal tibia. The left thigh can be cut with a bone cutter just above the knee. The forefoot can also be removed by cutting the distal tibia. Cut the skin laterally in ethanol and place the remainder of the forefoot in a 50 cc tube filled with 70% ethanol. The tube is stored at room temperature until transport. Rat tibia specimens are thawed to room temperature on the day of testing. The excised rat tibia is subjected to bone mineral density density measurements using peripheral quantitative computer-linked tomography (pQCT, XCT-RM, Norland / Stratec). Scans are performed at the proximal tibial site (12% of the total length away from the proximal end). Take one 0.5 mm slice. Scan as a whole (whole bone) or as an area that is 45% of the total bone tissue (cancellous bone) using experimental and internal boundary threshold illustrations (cortical bone) or by peeling away from the outer edge analyse. The bone mineral density, area and contents are then determined by the system software. Differences between sham and ovariectomized animals are determined at each different time point by two-tailed t-test using SAS statistical software (SAS Institute, Cory, NC). Student t test is used for statistical comparison of means. P values less than 0.05 are considered statistically significant.

(The activity of the albumin fusion protein encoded by construct 1941 can be assayed using an in vivo ovariectomized female rat model.)
The activity of the PTH albumin fusion protein encoded by construct 1941 can be measured using the in vivo assay described above under the title “In vivo ovariectomized female rat model”.

(Example 28: Construct ID 1949, HSA84-HSA, production)
Construct ID 1949 (pC4.HSA84.S1-Q84.HSA) encodes a PTH84-HSA fusion protein, which is an MPIF leader sequence followed by the amino form of the mature form of HSA cloned into the mammalian expression vector pC4. Includes the mature form of PTH84 (ie, Ser-1 to Gln-84) fused to the ends.

Cloning of PTH84 cDNA for construct 1949
DNA encoding PTH84 was amplified using the primers PTH84-3 and PTH84-4 described below, cut with BamHI / SpeI, and ligated into BamHI / XbaI cut pC4: HSA. Construct ID number 1949 encodes an albumin fusion protein comprising the mature PTH84 protein followed by the mature form of HSA.

  Two primers (PTH84-3 and PTH84-4) suitable for PCR amplification of polynucleotides encoding the mature form of PTH84 were synthesized.

ＰＴＨ８４−３は、Ｂａｍ−ＨＩクローニング部位（斜体で示される）、続いてＭＰＩＦシグナルペプチドをコードする核酸配列（下線を引いて示される）およびＰＴＨ８４の成熟形態のアミノ酸残基Ｓｅｒ−１〜Ｍｅｔ−８をコードする核酸配列（太字で示される）を含む。ＰＴＨ８４−４において、ＳｐｅＩ部位は、斜体で示され、そしてそれに続く核酸配列は、成熟ＰＴＨ８４タンパク質の最後の１０個のアミノ酸をコードするＤＮＡの逆相補体に対応する（太字で示される）。これらの２つのプライマーを使用して、ＰＴＨ８４タンパク質をＰＣＲ増幅した。ＰＣＲ増幅体を精製し、Ｂａｍ ＨＩおよびＳｐｅＩで消化し、そしてＢａｍ ＨＩ／ＸｂａＩ切断されたｐＣ４：ＨＳＡに連結した。

PTH84-3 is a Bam-HI cloning site (shown in italics) followed by the nucleic acid sequence encoding the MPIF signal peptide (shown underlined) and amino acid residues Ser-1 to Met- of the mature form of PTH84. Contains the nucleic acid sequence encoding 8 (shown in bold). In PTH84-4, the SpeI site is shown in italics, and the subsequent nucleic acid sequence corresponds to the reverse complement of DNA encoding the last 10 amino acids of the mature PTH84 protein (shown in bold). Using these two primers, the PTH84 protein was PCR amplified. The PCR amplification was purified, digested with Bam HI and Spe I, and ligated to Bam HI / Xba I cut pC4: HSA.

  As a result of the introduction of the SpeI cloning site into the PTH84-4 primer, two additional amino acid residues (ie, Thr and Ser) are present between PTH84 and HSA.

  Further analysis by amino acid sequencing of the N-terminus of the expressed albumin fusion protein confirmed the presence of the predicted PTH84 sequence (see below).

  The PTH84 albumin fusion protein of the invention is preferably a mature form of HSA (ie Asp-25) fused to either the N-terminus or C-terminus of the mature form of PTH84 (ie Ser-1 to Gln-84). ~ Leu-609). In one embodiment of the invention, the PTH84 albumin fusion protein of the invention further comprises a signal sequence that directs the nascent fusion polypeptide in the secretory pathway of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed and the mature PTH84 albumin fusion protein is secreted directly into the culture medium. The PTH84 albumin fusion protein of the present invention includes heterologous signal sequences (MAF, INV, Ig, Fibulin B, Clusterin, insulin-like growth factor binding protein 4, modified HSA leader sequences (including chimeric HSA / MAF leader sequences, Or other heterologous signal sequences known in the art, including but not limited to). In a preferred embodiment, the PTH84 albumin fusion protein of the invention comprises native PTH84. In a further preferred embodiment, the PTH84 albumin fusion protein of the present invention further comprises an N-terminal methionine residue. Polynucleotides (including fragments and / or variants) encoding these polypeptides are also encompassed by the present invention.

(Expression and purification of construct ID 1949)
(Expression in 293T cells)
Construct 1949 was transfected into 293T cells by methods known in the art (eg, lipofectamine transfection) and selected with 100 nM methotrexate (see Example 6). Expression levels were tested by immunoblot detection using anti-HSA serum as the primary antibody.

(Purification from 293T cell supernatant)
The 293T cell supernatant containing secreted PTH84-HSA fusion protein expressed in construct ID numbers 1949 to 293T cells was purified as described in Example 7. Specifically, initial capture was performed using anionic HQ-50 resin at pH 7.2, sodium phosphate buffer (25 mM Na ₂ HPO ₄ (pH 7.2)) and 0-0.5 M NaCl. Hydrophobic interaction chromatography using 16 column volumes of salt gradient elution followed by Phenyl 650M resin (Tosohaas) using salt gradient elution of 36 column volumes of 2.75-0M NaCl, pH 7.2. ("HIC"). Here, the sample had a final conductivity of 180 mS. The sample was concentrated using HQ Poros 50 resin and 0.15M NaCl incremental salt step elution. The final buffer composition consisted of 25 mM Na ₂ HPO ₄ +150 mM NaCl (pH 7.2). N-terminal sequencing yielded the amino terminal sequence of the mature form of PTH84 (ie, SVSEI, SEQ ID NO: 2145). An approximate MW protein of 78 kDa was obtained. A final yield of 0.32 mg protein per liter 293T cell supernatant was obtained.

(In vitro introduction of cyclic AMP in SaOS2 cells by albumin fusion protein encoded by construct 1949)
(result)
Purified HSA-PTH84 albumin fusion protein from 293T cells expressing construct 1949 was tested in the in vitro assay described in Example 27 under the subtitle “In vitro introduction of cyclic AMP in SaOS2 cells”. HSA-PTH84 induced an increase in intracellular cyclic AMP levels.

(The activity of the albumin fusion protein encoded by construct 1949 can be assayed using TPTX animals.)
The activity of the PTH albumin fusion protein encoded by construct 1949 was determined using the in vivo assay described in Example 27 under the TPTX animal, and subsection title “In Vivo: Induced Calcium Release in TPTX Animals”. Can be measured.

(The activity of the albumin fusion protein encoded by construct 1949 can be assayed using an in vivo ovariectomized female rat model.)
The activity of the PTH albumin fusion protein encoded by construct 1949 was determined using the in vivo assay described in Example 27 under the TPTX animal, and the subsection titled “In vivo Ovariectomized Female Rat Model” Can be measured.

(Example 29: Construct ID 2021, PTH84-HSA, production)
Construct ID 2021 (pC4.PTH84.S1-Q84.HSA) encodes a PTH84-HSA fusion protein, which is a native HSA leader sequence, followed by the mature form of HSA cloned into the mammalian expression vector pC4. Includes the mature form of PTH84 (ie, Ser-1 to Gln-84) fused to the amino terminus.

(Cloning of PTH84 cDNA for construct 2021)
DNA encoding PTH84 is amplified using primers PTH84-5 and PTH84-6 described below, cut with Xho I / Cla I, and ligated to Xho I / Cla I cut pC4: HSA did. Construct ID number 2021 encodes an albumin fusion protein comprising the mature PTH84 protein followed by the mature form of HSA (see Example 5).

  Two primers (PTH84-5 and PTH84-6) were synthesized that were suitable for PCR amplification of the polynucleotide encoding the mature form of PTH84.

ＰＴＨ８４−５は、Ｘｈｏ Ｉクローニング部位（斜体で示される）を組み込む。Ｘｈｏ Ｉ部位は、それに続く核酸配列（下線を引いて示される）と組み合わせられて、ＨＳＡのキメラシグナルペプチドの最後の４つのアミノ酸残基をコードする。太字の核酸配列は、ＰＴＨ８４の成熟形態のアミノ酸残基Ｓｅｒ−１〜Ａｓｎ−１０をコードする。ＰＴＨ８４−６において、Ｃｌａ Ｉ部位は、斜体で示され、そしてそれに続く核酸配列（下線を引いて示される）は、ＨＳＡの成熟形態の最初の１０個のアミノ酸をコードするＤＮＡの逆相補体に対応する。ＰＴＨ８４−６において太字で強調される核酸配列は、ＰＴＨ８４の成熟形態の最後の１１個のアミノ酸をコードするＤＮＡの逆相補体に対応する。これらの２つのプライマーを使用して、ＰＴＨ８４タンパク質をＰＣＲ増幅した。ＰＣＲ増幅体を精製し、Ｘｈｏ ＩおよびＣｌａ Ｉで消化し、そしてＸｈｏ Ｉ／Ｃｌａ Ｉ切断されたｐＣ４：ＨＳＡに連結した。

PTH84-5 incorporates a Xho I cloning site (shown in italics). The Xho I site, combined with the subsequent nucleic acid sequence (shown underlined), encodes the last four amino acid residues of the HSA chimeric signal peptide. The bold nucleic acid sequence encodes amino acid residues Ser-1 to Asn-10 of the mature form of PTH84. In PTH84-6, the Cla I site is shown in italics and the subsequent nucleic acid sequence (shown underlined) is the reverse complement of DNA encoding the first 10 amino acids of the mature form of HSA. Correspond. The nucleic acid sequence highlighted in bold in PTH84-6 corresponds to the reverse complement of DNA encoding the last 11 amino acids of the mature form of PTH84. Using these two primers, the PTH84 protein was PCR amplified. The PCR amplification was purified, digested with Xho I and Cla I, and ligated to Xho I / Cla I cut pC4: HSA.

  Furthermore, analysis by amino acid sequencing of the N-terminus of the expressed albumin fusion protein can confirm the presence of the predicted PTH84 sequence (see below).

  The PTH84 albumin fusion protein of the invention is preferably a mature form of HSA (ie Asp-25) fused to either the N-terminus or C-terminus of the mature form of PTH84 (ie Ser-1 to Gln-84). ~ Leu-609). In one embodiment of the invention, the PTH84 albumin fusion protein of the invention further comprises a signal sequence that directs the nascent fusion polypeptide in the secretory pathway of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed and the mature PTH84 albumin fusion protein is secreted directly into the culture medium. The PTH84 albumin fusion protein of the present invention includes heterologous signal sequences (MAF, INV, Ig, Fibulin B, Clusterin, insulin-like growth factor binding protein 4, modified HSA leader sequences (including chimeric HSA / MAF leader sequences, Or other heterologous signal sequences known in the art, including but not limited to). In a preferred embodiment, the PTH84 albumin fusion protein of the invention comprises native PTH84. In a further preferred embodiment, the PTH84 albumin fusion protein of the present invention further comprises an N-terminal methionine residue. Polynucleotides (including fragments and / or variants) encoding these polypeptides are also encompassed by the present invention.

(Expression and purification of construct ID2021)
(Expression in 293T cells)
Construct 2021 can be transfected into 293T cell cells by methods known in the art (eg, Lipofectamine transfection) and selected with 100 nM methotrexate (see Example 6). Expression levels can be tested by immunoblot detection using anti-HSA serum as the primary antibody.

(Purification from 293T cell supernatant)
The 293T cell supernatant containing the secreted PTH84-HSA fusion protein expressed from construct ID number 2021 in 293T cells can be purified as described in Example 7. In particular, if the sample has a final conductivity of 180 mS, initial capture was performed with anionic HQ-50 resin at pH 7.2 with sodium phosphate buffer (25 mM Na ₂ HPO ₄ (pH 7.2)) and 16 × Phenyl 650M resin (Tosohaas) using a column volume of 0-0.5M NaCl salt gradient elution followed by 36 column volumes of 2.75-0M NaCl salt gradient elution at pH 7.2. To) to perform a hydrophobic interaction chromatography, “HIC”. Samples can be concentrated using HQ Poros 50 resin and 0.15 mM increasing NaCl salt step elution. The final buffer composition can consist of 25 mM Na ₂ HPO ₄ +150 mM NaCl (pH 7.2). N-terminal sequencing should yield the amino terminal sequence of the mature form of PTH84 (ie, SVSEI). A protein with an approximate MW of 78 kDa should be obtained.

(In vitro induction of cyclic AMP in SaOS2 cells by albumin fusion protein encoded by construct 2021)
HSA-PTH84 albumin fusion protein derived from 293T cells expressing construct 2021 can be tested in the in vitro assay described in Example 27 under the title “In vitro induction of cyclic AMP in SaSO2 cells”.

(The activity of the albumin fusion protein encoded by construct 2021 can be assayed using TPTX animals)
The activity of the PTH albumin fusion protein encoded by construct 2021 can be measured using the TPTX animal and in vivo assays described in Example 27 under the title “In Vivo: Induced Release of Calcium in TPTX Animals”.

(The activity of the albumin fusion protein encoded by construct 2021 can be assayed using an in vivo ovariectomized female rat model)
The activity of the PTH albumin fusion protein encoded by construct 2021 can be measured using the in vivo assay described in Example 27 under the title “In vivo ovariectomized female rat model”.

(Example 30: Index for PTH84 albumin fusion protein)
The results from the in vitro and in vivo assays described above indicate that the PTH84 albumin fusion protein is useful for the treatment, prevention, and / or diagnosis of osteoporosis, malignant hypercalcemia, and Paget's disease.

(Example 31: Construction ID 2249 (IFNa2-HSA) production)
Construct ID 2249 (pSAC35: IFNa2.HSA) is designated as yeast S. cerevisiae. DNA encoding the IFNa2 albumin fusion protein with the HSA chimeric leader sequence followed by the mature form of IFNa2 protein (ie C1-E165) fused to the amino acid terminus of the mature form of HSA in the cerevisiae expression vector pSAC35.

(Cloning of IFNa2 cDNA)
The polynucleotide encoding IFNa2 was PCR amplified using the primers IFNa2-1 and IFNa2-2 described below. The PCR amplification was cut with SalI / ClaI and ligated to XhoI / ClaI cut sPcCHSA. Construct ID number 2249 encodes an albumin fusion protein comprising a chimeric leader sequence of HSA, a mature form of IFNa2, followed by a mature HSA protein.

  Two oligonucleotides suitable for PCR amplification of polynucleotides encoding mature forms IFNa2, IFNa2-1 and IFNa2-2 were synthesized;

ＩＦＮａ２−１プライマーは、ＳａｌＩクローニング部位（下線で示される）、キメラＨＳＡリーダー配列の最後の３個のアミノ酸残基をコードするヌクレオチド、およびＩＦＮａ２の成熟形態の最初の７個のアミノ酸残基をコードする２２ヌクレオチド（太字で示される）を組み込む。ＩＦＮａ２−２において、ＣｌａＩ部位（下線で示される）およびそれに続くＤＮＡは、成熟ＨＳＡタンパク質の最初の１０個のアミノ酸をコードするＤＮＡの逆相補鎖であり、そして最後の２２個のヌクレオチド（太字で示される）は、ＩＦＮａ２の最後の７個のアミノ酸をコードするＤＮＡの逆相補鎖である（実施例２を参照のこと）。ＩＦＮａ２−ＨＳＡのＰＣＲ増幅物をこれらのプライマーを使用して生成し、精製し、ＳａｌＩおよびＣｌａＩ制限酵素で消化し、そしてｐＳｃＣＨＳＡベクターのＸｈｏＩおよびＣｌａＩ部位でクローニングした。配列を確認した後、このＩＦＮａ２アルブミン融合タンパク質をコードする発現カセットはＮｏｔＩで消化したｐＳＡＣ３５にサブクローニングした。

The IFNa2-1 primer encodes the SalI cloning site (shown underlined), the nucleotide encoding the last 3 amino acid residues of the chimeric HSA leader sequence, and the first 7 amino acid residues of the mature form of IFNa2. 22 nucleotides (shown in bold). In IFNa2-2, the ClaI site (indicated by underlining) followed by DNA is the reverse complementary strand of DNA encoding the first 10 amino acids of the mature HSA protein and the last 22 nucleotides (in bold) (Shown) is the reverse complementary strand of DNA encoding the last 7 amino acids of IFNa2 (see Example 2). A PCR amplification of IFNa2-HSA was generated using these primers, purified, digested with SalI and ClaI restriction enzymes, and cloned at the XhoI and ClaI sites of the pScCHSA vector. After confirming the sequence, the expression cassette encoding this IFNa2 albumin fusion protein was subcloned into NotI digested pSAC35.

  Furthermore, analysis of the N-terminus of the expressed albumin fusion protein by amino acid sequencing can confirm the presence of the expected IFNa2 sequence (see below).

  Other IFNa2 albumin fusion proteins that use different leader sequences have been constructed by methods known in the art (see Example 2). Examples of various leader sequences include, but are not limited to, invertase “INV” (constructs 2343 and 2410) and mating mating alpha factor “MAF” (construct 2366) These IFNa2 albumin fusion proteins were previously Can be subcloned into mammalian expression vectors such as pC4 (construct 2382) and pEE12.1 as described (see Example 5) IFNa2 albumin fusion protein with a therapeutic moiety fused C-terminal to HSA Can also be constructed (construct 2381).

  The IFNa2 albumin fusion protein of the present invention is preferably a mature form of HSA (ie Asp-25) fused to either the N-terminus or C-terminus of the mature form of IFNa2 (ie Cys-1 to Glu-165). ~ Leu-609). In one embodiment of the invention, the IFNa2 albumin fusion protein of the invention further comprises a signal sequence that directs the initial (early) fusion polypeptide to the secretory pathway of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed and the mature IFNa2 albumin fusion protein is secreted directly into the culture medium. The IFNa2 albumin fusion protein of the present invention comprises a heterologous signal sequence (MAF, INV, Ig, fibrin B, clusterin, insulin-like growth factor binding protein 4, HSA leader sequence variant (chimeric HSA / MAF) known in the art. Including, but not limited to, leader sequences) or other heterologous signal sequences. In a preferred embodiment, the IFNa2 albumin fusion protein of the invention comprises native IFNa2. In a further preferred embodiment, the IFNa2 albumin fusion protein of the invention further comprises an N-terminal methionine residue. Polynucleotides encoding these polypeptides (including fragments and / or variants) are also encompassed by the present invention.

(Expression and purification of construct ID 2249)
(Expression in yeast S. cerevisiae)
Construct 2249, yeast S. cerevisiae. Transformation with cerevisiae strain BXP10 was performed by methods known in the art (see Example 3). Cells can be harvested in stationary phase after 72 hours of growth. The supernatant is collected by removing the impurities at 3000 g for 10 minutes. Expression levels can be tested with anti-HSA serum (Kent Laboratories) or by immunoblot detection as this primary antibody. An IFNa2 albumin fusion protein with a molecular weight of approximately 88.5 kDa can be recovered.

(Purification from the supernatant of yeast S. cerevisiae cells)
Yeast S. Cell supernatant containing IFNa2 albumin fusion protein expressed from construct ID number 2249 in cerevisiae cells on a Dyax peptide affinity column on a small scale (see Example 4) or the following five steps: Diafiltration , Anion exchange chromatography using DEAE-Sepharose Fast Flow column, hydrophobic interaction chromatography (HIC) using Butyl 650S column, cation exchange using SP-Sepharose Fast Flow column or Blue-Sepharose chromatography Chromatography and high performance chromatograph using Q-sepharose high performance column chromatography It can be purified either on a large scale by Fee (see Example 4). IFNa2 albumin fusion protein is eluted from DEAE-Sepharose Fast Flow column at 100-250 mM NaCl, from SP-Sepharose Fast Flow column at 150-250 mM NaCl, and from Q-Sepharose High Performance column at 5-7.5 mS / cm. obtain. N-terminal sequencing should yield the sequence CDLPQ (SEQ ID NO: 2146) corresponding to the mature form of IFNa2.

(IFNa2 activity can be assayed using in vitro ISRE-SEAP assay)
(Method)
The IFNa2 albumin fusion protein encoded by construct ID number 2249 can be tested for activity in an ISRE-SEAP assay as previously described in Example 25. Briefly, conditioned yeast supernatants were tested at a 1: 1000 dilution for the ability to direct ISRE signaling against the ISRE-SEAP / 293F reporter cell line. ISRE-SEAP / 293F reporter cells were plated in 96-well poly-D-lysine coated plates at 3 × 10 ⁴ cells / well 1 day prior to treatment. The reporter cells were then incubated for 18 or 24 hours before removing 40 μL for use in the SEAP Reporter Gene Chemiluminescent Assay (Roche catalog number 1779842). Recombinant human Interferon β “rhIFNb” (Biogen) was used as a positive control.

(result)
Purified preparations of IFNa2-HSA are 10 ⁻¹ to 10 ¹ ng / mL (see FIG. 15) or 10 ⁻¹⁰ to 10 ⁻⁸ ng / mL (see FIG. 16) in the ISRE-SEAP assay. A relatively linear increase over concentrations in the range of

(In vivo induction of OAS by interferon alpha fusion encoded by construct ID 2249)
(Method)
The OAS enzyme (2′-5′-oligoadenylate synthetase) is activated at the transcriptional level by interferon in response to antiviral infection. The effect of interferon assembly can be measured by collecting blood samples from treated monkeys and analyzing these samples for the transcriptional activity of two OAS mRNAs (p41 and p69). A 0.5 mL volume of whole blood was collected from 4 animals per group at 7 different time points (0 animal, 1 day, 2 days, 4 days, 8 days, 10 animals, 10 animals per animal). Day and day 14). The various groups consisted of vehicle control (intravenous injection of 30 μg / kg HSA-IFN on day 1, subcutaneous injection of 30 μg / kg HSA-IFN on day 1, and 300 μg / kg HSA-IFN on day 1. Subcutaneous injection) and as a positive control include subcutaneous injection of 40 μg / kg of interferon α (Schering-Plough) on days 1, 3, and 5. The levels of p41 and p69 mRNA transcripts were determined by real-time quantitative PCR (Taqman) using probes specific for p41-OAS and p69-OAS. OAS mRNA levels were quantified compared to the endogenous control of 18S ribosomal RNA. The albumin fusion protein encoded by construct 2249 can be subjected to similar experiments.

(result)
Significant increases in mRNA transcript levels for both p41OAS and p69OAS were observed in monkeys treated with HSA-interferon as opposed to monkeys treated with IFNa (see FIG. 17 for p41 data). The effect lasted nearly 10 days.

(Example 32: Index for IFNa2 albumin fusion protein)
IFNα albumin fusion proteins (including those encoded by constructs 2249, 2343, 2410, 2366, 2382, and 2381 to treat, prevent, ameliorate, and / or detect multiple sclerosis, including Non-limiting) may be used. Other indicators include hepatitis C, use in oncology, cancer, hepatitis, human papillomavirus, fibromyomatosis, Sjogren's syndrome, hairy cell leukemia, chronic myelogenous leukemia, AIDS-related capage sarcoma, chronic hepatitis B, Malignant melanoma, non-Hodgkin lymphoma, external wart condylome, HIV infection, small cell lung cancer, hematological malignancy, herpes simplex virus infection, multiple sclerosis, viral hemorrhagic fever, solid tumor, kidney cancer, bone marrow Injuries, bone injuries, bladder cancer, gastric cancer, hepatitis D, multiple myeloma, type I diabetes, viral infection, cutaneous T-cell lymphoma, cervical dysplasia, chronic fatigue syndrome, and kidney cancer. It is not limited to.

  Preferably, the IFNα-albumin fusion protein or IFN hybrid fusion protein is in combination with a CCR5 antagonist, and in addition to at least one ribavirin, IL-2, IL-12, untreated adult patients and children For the preparation of a medicament for the treatment of HIV-1 infection, HCV, or co-infection of HIV-1 and HCV in patients and treated adult and pediatric patients, pentafside alone or anti Administered in combination with HIV drug therapy (eg, HAART).

(Example 33: Construction ID2250 (HSA-insulin (GYG)) production)
Construct ID 2250 (pSAC35.HSA.INSULIN (GYG) .F1-N62) is designated as yeast S. cerevisiae. A full-length HSA (including native HSA leader sequence) fused to the amino terminus of a synthetic single-chain, long-acting insulin analogue (INSULIN (GY ³² G)) at position 32 Tyr cloned into the cerevisiae expression vector pSAC35 Which encodes an HSA-INSULIN (GYG) fusion protein.

(Cloning of INSULIN (GYG) cDNA for Construct 2250)
DNA encoding a single synthetic form of INSULIN (GYG) was PCR synthesized using four overlapping primers. To eliminate the need for proinsulin processing and ensure correct single-stranded protein folding, the sequence corresponding to the C peptide in the intermediate region of the proinsulin cDNA was designated as insulin growth factor 1 ("IGF- 1 ") C domain (GY ³² GSSSRRAPQT, SEQ ID NO: 2147). The sequence is yeast S. cerevisiae. Codon optimized for expression in C. cerevisiae. This PCR fragment was digested and subcloned into pScNHSA digested with Bsu361 / AscI. The NotI fragment was then subcloned into the pSAC35 plasmid. Construct ID number 2250 encodes full length HSA (including the native HSA leader sequence) fused to the amino terminus of the synthetic single-stranded form of INSULIN (GYG).

  Four overlapping oligonucleotide 5 'and 3' primers suitable for PCR amplification of polynucleotides encoding synthetic single-stranded forms of INSULIN (GYG), INSULIN (GYG) -1 and INSULIN (GYG) -2 Synthesized:

ＩＮＳＵＬＩＮ（ＧＹＧ）−１は、Ｂｓｕ３６Ｉクローニング部位（イタリック体で示される）を組み込み、そしてＩＮＳＵＬＩＮ（ＧＹＧ）の合成一本鎖形態のＯＲＦの最初の２１個のアミノ酸（太字で示される）をコードする。ＩＮＳＵＬＩＮ（ＧＹＧ）−２において、イタリック体にした配列は、ＡｓｃＩ部位である。ＩＮＳＵＬＩＮ（ＧＹＧ）−２において、太字にされた配列は、ＩＮＳＵＬＩＮ（ＧＹＧ）の合成一本鎖形態のアミノ酸残基Ｃｙｓ−４９〜Ａｓｎ−６３をコードする最後の４９個のヌクレオチドの逆相補鎖である。これらの２つのプライマーで、ＩＮＳＵＬＩＮ（ＧＹＧ）の合成一本鎖形態をＰＣＲ増幅した。それぞれ特異的なプライマー対およびテンプレートについてのアニーリング温度および時間ならびに伸長温度および時間を、経験的に決定しなければならない。

INSULIN (GYG) -1 incorporates a Bsu36I cloning site (shown in italics) and encodes the first 21 amino acids (shown in bold) of the ORS in a synthetic single-chain form of INSULIN (GYG) . In INSULIN (GYG) -2, the italicized sequence is the AscI site. In INSULIN (GYG) -2, the bolded sequence is the reverse complementary strand of the last 49 nucleotides encoding amino acid residues Cys-49 to Asn-63 in the synthetic single-stranded form of INSULIN (GYG). is there. With these two primers, a synthetic single-stranded form of INSULIN (GYG) was PCR amplified. The annealing temperature and time and extension temperature and time for each specific primer pair and template must be determined empirically.

  The PCR product was purified (eg, using the Wizard PCR Preps DNA Purification System (Promega Corp)) and then digested with Bsu36I and AscI. After further purification of the Bsu36I-AscI fragment by gel electrophoresis, the product was cloned into pScNHSA digested with Bsu36I / AscI. Furthermore, the NotI fragment was subcloned into pSAC35, resulting in construct ID number 2250.

  Further analysis of the N-terminus of the expressed albumin fusion protein by amino acid sequencing should confirm the presence of the expected mature HSA sequence (see below).

The INSULIN albumin fusion protein of the present invention is preferably a mature form of HSA (ie Asp The sequence corresponding to the C peptide in the intermediate region of the proinsulin cDNA is replaced by the C domain of insulin growth factor 1 (“IGF-1”) (GY ³² GSSRRAPQT, SEQ ID NO: 2147). did. In one embodiment of the invention, the INSULIN albumin fusion protein of the invention further comprises a signal sequence that directs the initial fusion polypeptide into the secretory pathway of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed and the mature INSULIN albumin fusion protein is secreted directly into the culture medium. The INSULIN albumin fusion protein of the present invention includes heterologous signal sequences (MAF, INV, Ig, fibrin B, clusterin, insulin-like growth factor binding protein 4, HSA leader sequence variants (including chimeric HSA / MAF leader sequences, But not limited thereto) or other heterologous signal sequences known in the art. In a preferred embodiment, the INSULIN albumin fusion protein of the invention comprises native INSULIN. In a further preferred embodiment, the INSULIN albumin fusion protein of the present invention further comprises an N-terminal methionine residue. Polynucleotides encoding these polypeptides (including fragments and / or variants) are also encompassed by the present invention.

(Expression and purification of construct ID2250)
(Expression in yeast S. cerevisiae)
Construct 2250 is obtained from yeast S. coli by methods known in the art. can be transformed with C. cerevisiae (see Example 3). Expression levels can be tested by immunoblot detection using anti-HSA serum as the primary antibody.

(Purification from the supernatant of yeast S. cerevisiae cells)
Yeast S. In C. cerevisiae, the cell supernatant containing the secreted INSULIN (GYG) albumin fusion protein expressed from construct ID number 2250 can be purified as described in Example 4. N-terminal sequencing of this albumin fusion protein should yield the sequence DAHKS (SEQ ID NO: 2143) corresponding to the amino terminus of the mature form of HSA.

In vitro [ ³ H] -2-deoxyglucose uptake assay in the presence of albumin fusion protein encoded by construct 2250
(Method)
An in vitro assay to measure sugar uptake in 3T3-L1 adipocytes in the presence of INSULIN (GYG) albumin fusion protein encoded by construct 2250 was performed as described below in Example 41. Other assays known in the art that can be used to test INSULIN (GYG) albumin fusion proteins include L6 rat myoblast proliferation assay via glycogen synthesis kinase-3 (GSK-3) and rat malate dehydrogenase HAIIe reporter assay comprising promoter (rMEP) -SEAP reporter, sterol regulatory element binding protein (SREBP) -SEAP reporter, fatty acid synthesis (FAS) -SEAP reporter, and phosphoenolpyruvate carboxykinase (PEPCK) -SEAP reporter (Examples) 48)), but is not limited thereto.

(result)
Transformed yeast expressing the insulin albumin fusion protein encoded by construct 2250 The supernatant from C. cerevisiae demonstrated sugar uptake / transport activity in 3T3-L1 adipocytes (see FIG. 18).

In vitro pancreatic cell line proliferation assay in the presence of albumin fusion protein encoded by construct 2250
(Method)
Measure the differentiation and proliferation of ductal epithelial pancreatic ARIP cell line into insulin producing β cells in the presence of INSULIN (GYG) albumin fusion protein encoded by construct 2250 and / or the growth of insulin producing RIN-Mβ cell line An in vitro assay to measure can be performed as described below under the heading "Example 42: In vitro assay of [ ³ H] thymidine incorporation into pancreatic cell lines".

(The activity of the albumin fusion protein encoded by construct 2250 can be assayed in vivo using a diabetic NOD and / or NIDDM mouse model)
The activity of the INSULIN (GYG) albumin fusion protein encoded by construct 2250 was determined under the headings "Example 44: Development of diabetes in NOD mice", "Example 45: Histological experiments in NOD mice", and "Example 47. Can be measured using the NOD and / or NIDDM mouse model described below under “: In vivo mouse model of NIDDM”.

(Example 34: Construct ID 2255 (insulin (GYG) -HSA) production)
Construct ID 2255 (pSAC35.INSULIN (GYG) .F1-N62.HSA) is designated as yeast S. cerevisiae. A Tyr at position 32 cloned in the cerevisiae expression vector pSAC35 is fused to the amino terminus of a synthetic single-chain, long-acting insulin analog (INSULIN (GY ³² G)), which is then fused to the mature form of HSA. The HSA chimeric leader sequence of HSA is encoded and encodes an INSULIN (GYG) -HSA fusion protein.

(Cloning of INSULIN (GYG) cDNA for Construct 2255)
DNA encoding a synthetic single-stranded form of INSULIN (GYG) was PCR generated using four overlapping primers. To eliminate the need for proinsulin processing and ensure correct single-stranded protein folding, the sequence corresponding to the C peptide in the intermediate region of the proinsulin cDNA was insulin growth factor 1 ("IGF-1 )) C domain (GY ³² GSSSRRAPQT, SEQ ID NO: 2147). The sequence is yeast S. cerevisiae. Codon optimized for expression in C. cerevisiae. This PCR fragment was digested with SalI / ClaI and subcloned into pScCHSA digested with XhoI / ClaI. The NotI fragment was then subcloned into the pSAC35 plasmid. Construct ID number 2250 encodes the HSA chimeric leader sequence fused to the amino terminus of the synthetic single-stranded form of INSULIN (GYG) following the mature form of HSA.

  Four overlapping oligonucleotide 5 ′ and 3 ′ primers suitable for PCR amplification of polynucleotides encoding synthetic single-stranded forms of INSULIN (GYG), INSULIN (GYG) -3, and INSULIN (GYG) -4 Synthesized:

ＩＮＳＵＬＩＮ（ＧＹＧ）−３は、ＳａｌＩクローニング部位（イタリック体で示される）およびＩＮＳＵＬＩＮ（ＧＹＧ）の合成一本鎖形態のＯＲＦの最初の２１個のアミノ酸（太字で示される）をコードするＤＮＡを組み込む。ＩＮＳＵＬＩＮ（ＧＹＧ）−４において、イタリック体化された配列は、ＣｌａＩ部位であり；そしてこのＣｌａＩ部位およびそれに続くＤＮＡは、成熟ＨＳＡタンパク質の最初の１０個のアミノ酸をコードするＤＮＡの逆相補鎖である。太字にされた配列は、ＩＮＳＵＬＩＮ（ＧＹＧ）の合成一本鎖形態の最後の１５個のアミノ酸残基Ｃｙｓ−４９〜Ａｓｎ−６３をコードする４６個のヌクレオチドの逆相補鎖である。これらの２つのプライマーで、ＩＮＳＵＬＩＮ（ＧＹＧ）の合成一本鎖形態を、アニーリング、アニーリングされたプライマーの伸長、ＳａｌＩおよびＣｌａＩでの消化、およびＸｈｏＩ／ＣｌａＩで消化したｐＳｃＣＨＳＡへのサブクローニングによって生成した。次いで、このクローン由来のＮｏｔＩフラグメントをｐＳＡＣ３５のＮｏｔＩ部位にライゲーションして、構築物ＩＤ２２５５を生成した。構築物ＩＤ番号２２５５は、キメラリーダー配列、ＩＮＳＵＬＩＮ（ＧＹＧ）の合成一本鎖、およびＨＳＡの成熟形態を含むアルブミン融合タンパク質をコードする。

INSULIN (GYG) -3 incorporates DNA encoding the SalI cloning site (shown in italics) and the first 21 amino acids (shown in bold) of the synthetic single-stranded form of INSULIN (GYG) . In INSULIN (GYG) -4, the italicized sequence is a ClaI site; and this ClaI site and subsequent DNA are the reverse complementary strand of DNA encoding the first 10 amino acids of the mature HSA protein. is there. The bolded sequence is a 46 nucleotide reverse complementary strand encoding the last 15 amino acid residues Cys-49-Asn-63 in a synthetic single-stranded form of INSULIN (GYG). With these two primers, a synthetic single-stranded form of INSULIN (GYG) was generated by annealing, extension of the annealed primer, digestion with SalI and ClaI, and subcloning into pScCHSA digested with XhoI / ClaI. The NotI fragment from this clone was then ligated to the NotI site of pSAC35 to generate construct ID 2255. Construct ID number 2255 encodes an albumin fusion protein comprising a chimeric leader sequence, a synthetic single chain of INSULIN (GYG), and a mature form of HSA.

  Further analysis of the N-terminus of the expressed albumin fusion protein by amino acid sequencing should confirm the presence of the expected INSULIN (GYG) sequence (see below).

The INSULIN albumin fusion protein of the present invention is preferably a mature form of HSA (ie Asp The sequence corresponding to the C peptide in the intermediate region of the proinsulin cDNA is replaced by the C domain of insulin growth factor 1 (“IGF-1”) (GY ³² GSSRRAPQT, SEQ ID NO: 2147). did. In one embodiment of the invention, the INSULIN albumin fusion protein of the invention further comprises a signal sequence that directs the initial fusion polypeptide into the secretory pathway of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed and the mature INSULIN albumin fusion protein is secreted directly into the culture medium. The INSULIN albumin fusion protein of the present invention includes heterologous signal sequences (MAF, INV, Ig, fibrin B, clusterin, insulin-like growth factor binding protein 4, HSA leader sequence variants (including chimeric HSA / MAF leader sequences, But not limited thereto) or other heterologous signal sequences known in the art. In a preferred embodiment, the INSULIN albumin fusion protein of the invention comprises native INSULIN. In a further preferred embodiment, the INSULIN albumin fusion protein of the present invention further comprises an N-terminal methionine residue. Polynucleotides encoding these polypeptides (including fragments and / or variants) are also encompassed by the present invention.

(Expression and purification of construct ID 2255)
(Expression in yeast S. cerevisiae)
Construct 2255 is obtained from yeast S. cerevisiae by methods known in the art. can be transformed with C. cerevisiae (see Example 3). Expression levels can be tested by immunoblot detection using anti-HSA serum as the primary antibody.

(Purification from the supernatant of yeast S. cerevisiae cells)
Yeast S. In C. cerevisiae, the cell supernatant containing the secreted INSULIN (GYG) albumin fusion protein expressed from construct ID number 2255 can be purified as described in Example 4. N-terminal sequencing of this expressed and purified albumin fusion protein should yield FVNQH corresponding to the amino terminus of a long acting insulin analog (INSULIN (GY ³² G)) in a synthetic single chain form.

In vitro [ ³ H] -2-deoxyglucose uptake assay in the presence of albumin fusion protein encoded by construct 2255
(Method)
An in vitro assay to measure sugar uptake in 3T3-L1 adipocytes in the presence of INSULIN (GYG) albumin fusion protein encoded by construct 2255 was performed as described below in Example 41. Other assays known in the art that can be used to test INSULIN (GYG) albumin fusion proteins include L6 rat myoblast proliferation assay via glycogen synthesis kinase-3 (GSK-3) and rat malate enzyme H4IIe reporter assay comprising promoter (rMEP) -SEAP reporter, sterol regulatory element binding protein (SREBP) -SEAP reporter, fatty acid synthesis (FAS) -SEAP reporter, and phosphoenolpyruvate carboxykinase (PEPCK) -SEAP reporter (Examples) 48)), but is not limited thereto.

In vitro pancreatic cell line proliferation assay in the presence of albumin fusion protein encoded by construct 2255
(Method)
Measure the differentiation and proliferation of ductal epithelial pancreatic ARIP cell line into insulin-producing β cells in the presence of INSULIN (GYG) albumin fusion protein encoded by construct 2255 and / or An in vitro assay to measure can be performed as described below under the heading "Example 42: In vitro assay of [ ³ H] thymidine incorporation into pancreatic cell lines".

(The activity of the albumin fusion protein encoded by construct 2255 may be assayed in vivo using a diabetic NOD and / or NIDDM mouse model)
The activity of the INSULIN (GYG) albumin fusion protein encoded by construct 2255 was determined using the titles “Example 44: Development of diabetes in NOD mice”, “Example 45: Histological experiments in NOD mice”, and “Example 47”. Can be measured using the NOD and / or NIDDM mouse model described below under “: In vivo mouse model of NIDDM”.

Example 35: Generation of construct ID 2276 (HSA-insulin (GGG))
Construct ID 2276 (pSAC35.HSA.INSULIN (GGG) .F1-N58) is designated as yeast S. cerevisiae. Full-length HSA (including native HSA leader sequence) fused to the amino terminus of a Gly, synthetic single-chain, long-acting insulin analog (INSULIN (GG ³² G)) at position 32 cloned into the cerevisiae expression vector pSAC35 HSA-INSULIN (GGG) fusion protein is encoded.

(Cloning of ISULIN (GGG) cDNA for construct 2276)
DNA encoding a synthetic single-stranded form of INSULIN (GGG) was PCR generated using four overlapping primers. To eliminate the need for proinsulin processing and to ensure correct single-stranded protein folding, a sequence corresponding to the C peptide in the intermediate region of the proinsulin cDNA is a synthetic linker ("GG ³² GPGKR"). Replaced by (SEQ ID NO: 2148). The sequence is yeast S. cerevisiae. Codon optimized for expression in C. cerevisiae. This PCR fragment was digested and subcloned into pScNHSA digested with Bsu361 / AscI. The NotI fragment was then subcloned into the pSAC35 plasmid. Construct ID number 2276 encodes full-length HSA containing the native HSA leader sequence fused to the amino terminus of the synthetic single-stranded form of INSULIN (GGG).

  Four overlapping oligonucleotide 5 ′ and 3 ′ primers suitable for PCR amplification of polynucleotides encoding synthetic single-stranded forms of INSULIN (GGG), INSULIN (GGG) -1 and INSULIN (GGG) -2 Synthesized:

ＩＮＳＵＬＩＮ（ＧＧＧ）−５は、Ｂｓｕ３６Ｉクローニング部位（イタリック体で示される）を組み込み、そしてＩＮＳＵＬＩＮ（ＧＧＧ）の合成一本鎖形態のＯＲＦの最初の２１個のアミノ酸（太字で示される）をコードする。ＩＮＳＵＬＩＮ（ＧＧＧ）−６において、イタリック体化された配列は、ＡｓｃＩ部位である。ＩＮＳＵＬＩＮ（ＧＧＧ）−６において、太字にされた配列は、ＩＮＳＵＬＩＮ（ＧＧＧ）の合成一本鎖形態のアミノ酸残基Ｃｙｓ−４４〜Ａｓｎ−５８をコードする最後の４９個のヌクレオチドの逆相補鎖である。これらの２つのプライマーで、ＩＮＳＵＬＩＮ（ＧＧＧ）の合成一本鎖形態をＰＣＲ増幅した。それぞれ特異的なプライマー対およびテンプレートについてのアニーリング温度および時間ならびに伸長温度および時間を、経験的に決定しなければならない。

INSULIN (GGG) -5 incorporates the Bsu36I cloning site (shown in italics) and encodes the first 21 amino acids (shown in bold) of the ORS in the synthetic single-chain form of INSULIN (GGG) . In INSULIN (GGG) -6, the italicized sequence is the AscI site. In INSULIN (GGG) -6, the bolded sequence is the reverse complementary strand of the last 49 nucleotides encoding amino acid residues Cys-44 to Asn-58 in the synthetic single-stranded form of INSULIN (GGG). is there. With these two primers, a synthetic single-stranded form of INSULIN (GGG) was PCR amplified. The annealing temperature and time and extension temperature and time for each specific primer pair and template must be determined empirically.

  The PCR product was purified (eg, using the Wizard PCR Preps DNA Purification System (Promega Corp)) and then digested with Bsu36I and AscI. After further purification of the Bsu36I-AscI fragment by gel electrophoresis, the product was cloned into pScNHSA digested with Bsu36I / AscI. The NotI fragment was further subcloned into pSAC35 to provide construct ID number 2276.

  Further analysis of the N-terminus of the expressed albumin fusion protein by amino acid sequencing should confirm the presence of the expected mature HSA sequence (see below).

The INSULIN albumin fusion protein of the present invention is preferably a mature form of HSA (ie Asp-25 to Leu-609); the sequence corresponding to the C peptide in the middle region of the proinsulin cDNA was replaced by a synthetic linker (“GG ³² GPGKR”) (SEQ ID NO: 2148). In one embodiment of the invention, the INSULIN albumin fusion protein of the invention further comprises a signal sequence directed to the starting fusion polypeptide in the host secretory pathway used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed and the mature INSULIN albumin fusion protein is secreted directly into the culture medium. The INSULIN albumin fusion protein of the present invention comprises a heterologous signal sequence known in the art (MAF, INV, Ig, fibrin B, clusterin, insulin-like growth factor binding protein 4, HSA leader sequence variant (chimeric HSA / MAF leader sequence Including, but not limited to), or other heterologous signal sequences. In a preferred embodiment, the INSULIN albumin fusion protein of the invention comprises native INSULIN. In a further preferred embodiment, the INSULIN albumin fusion protein of the present invention further comprises an N-terminal methionine residue. Polynucleotides encoding these polypeptides (including fragments and / or variants) are also encompassed by the present invention.

(Expression and purification of construct ID 2276)
(Expression in yeast S. cerevisiae)
Construct 2276 is obtained from yeast S. cerevisiae by methods known in the art (see Example 3). cerevisiae can be transformed. Expression levels can be tested by immunoblot detection using anti-HSA serum as the primary antibody.

(Purification from the supernatant of yeast S. cerevisiae cells)
Yeast S. In C. cerevisiae, the cell supernatant containing the secreted INSULIN (GGG) albumin fusion protein expressed from construct ID number 2276 can be purified as described in Example 4. N-terminal sequencing should yield DAHKS (SEQ ID NO: 2143) corresponding to the amino terminus of the mature form of HSA.

In vitro [ ³ H] -2-deoxyglucose uptake assay in the presence of albumin fusion protein encoded by construct 2276
(Method)
An in vitro assay to measure sugar uptake in 3T3-L1 adipocytes in the presence of the INSULIN (GGG) albumin fusion protein encoded by construct 2276 was performed as described below in Example 41. Other assays known in the art that can be used to test INSULIN (GGG) albumin fusion proteins include L6 rat myoblast proliferation assay via glycogen synthesis kinase-3 (GSK-3) and rat malate dehydrogenase HAIIe reporter assay comprising promoter (rMEP) -SEAP reporter, sterol regulatory element binding protein (SREBP) -SEAP reporter, fatty acid synthase (FAS) -SEAP reporter, and phosphoenolpyruvate carboxykinase (PEPCK) -SEAP reporter See Example 48), but is not limited thereto.

(result)
Transformed yeast expressing the insulin albumin fusion encoded by construct 2276 The supernatant from C. cerevisiae demonstrated glucose uptake / transport activity in 3T3-L1 adipocytes (see FIG. 18).

In vitro pancreatic cell line proliferation assay in the presence of albumin fusion protein encoded by construct 2276
(Method)
Measure the differentiation and proliferation of ductal epithelial pancreatic ARIP cell line to insulin-producing β cells in the presence of INSULIN (GGG) albumin fusion protein encoded by construct 2276 and / or the growth of insulin-producing RIN-Mβ cell line An in vitro assay to measure can be performed as described below under the heading "Example 42: In vitro assay of [ ³ H] thymidine incorporation into pancreatic cell lines".

(The activity of the albumin fusion protein encoded by construct 2276 may be assayed in vivo using a diabetic NOD and / or NIDDM mouse model)
The activity of the INSULIN (GGG) albumin fusion protein encoded by construct 2276 was determined using the titles “Example 44: Development of diabetes in NOD mice”, “Example 45: Histological examination of NOD mice”, and “Example 47”. Can be measured using the NOD and / or NIDDM mouse model described below under “: In vivo mouse model of NIDDM”.

Example 36 Production of Construct ID 2278 (Insulin (GGG) -HSA)
Construct ID 2278 (pSAC35.INSULIN (GGG) .HSA) is designated as yeast S. cerevisiae. fused to the amino terminus of a Gly, synthetic single-chain, long-acting insulin analog (INSULIN (GG ³² G)) at position 32, containing the HSA chimera leader sequence of HSA cloned into the cerevisiae expression vector pSAC35, It is then fused to the mature form of HSA, encoding the INSULIN (GGG) -HSA fusion protein.

(Cloning of INSULIN (GGG) cDNA for Construct 2278)
DNA encoding a synthetic single-stranded form of INSULIN (GGG) was PCR generated using four overlapping primers. To eliminate the need for proinsulin processing and ensure correct single-stranded protein folding, sequences corresponding to the C peptide in the middle region of the proinsulin cDNA were synthesized with a synthetic linker ("GG ³² GPGKR" ) (SEQ ID NO: 2148). The sequence is yeast S. cerevisiae. Codon optimized for expression in C. cerevisiae. This PCR fragment was digested with SalI / ClaI and subcloned into pScCHSA digested with XhoI / ClaI. The NotI fragment was then subcloned into the pSAC35 plasmid. Construct ID number 2278 encodes the chimeric leader sequence of HSA fused to the amino terminus of the synthetic single-stranded form of INSULIN (GGG) followed by the mature form of HSA.

  Four overlapping oligonucleotide 5 'and 3' primers suitable for PCR amplification of polynucleotides encoding synthetic single-stranded forms of INSULIN (GGG), INSULIN (GGG) -7 and INSULIN (GGG) -8 Synthesized:

ＩＮＳＵＬＩＮ（ＧＧＧ）−７は、ＳａｌＩクローニング部位（イタリック体で示される）およびＩＮＳＵＬＩＮ（ＧＧＧ）の合成一本鎖形態のＯＲＦの最初の２１個のアミノ酸（太字で示される）をコードするＤＮＡを組み込む。ＩＮＳＵＬＩＮ（ＧＧＧ）−８において、イタリック体化された配列は、ＣｌａＩ部位であり；そしてこのＣｌａＩ部位およびそれに続くＤＮＡは、成熟ＨＳＡタンパク質の最初の１０個のアミノ酸をコードするＤＮＡの逆相補鎖である。太字にされた配列は、ＩＮＳＵＬＩＮ（ＧＧＧ）の合成一本鎖形態の最後の１５個のアミノ酸残基Ｃｙｓ−４４〜Ａｓｎ−５８をコードする４６個のヌクレオチドの逆相補鎖である。これらの２つのプライマーで、ＩＮＳＵＬＩＮ（ＧＧＧ）の合成一本鎖形態を、アニーリングし、アニーリングされたプライマーの伸長、ＳａｌＩおよびＣｌａＩでの消化、およびＸｈｏＩ／ＣｌａＩで消化したｐＳｃＣＨＳＡへのサブクローニングによって生成した。次いで、構築物ＩＤ２２７８を生成するために、このクローン由来のＮｏｔＩフラグメントを、ｐＳＡＣ３５のＮｏｔＩ部位にライゲーションした。構築物ＩＤ番号２２７８は、キメラリーダー配列、ＩＮＳＵＬＩＮ（ＧＧＧ）の合成一本鎖、およびＨＳＡの成熟形態を含むアルブミン融合タンパク質をコードする。

INSULIN (GGG) -7 incorporates DNA encoding the SalI cloning site (shown in italics) and the first 21 amino acids (shown in bold) of the synthetic single-stranded form of INSULIN (GGG) . In INSULIN (GGG) -8, the italicized sequence is a ClaI site; and this ClaI site and subsequent DNA are the reverse complementary strand of DNA encoding the first 10 amino acids of the mature HSA protein. is there. The bolded sequence is a 46 nucleotide reverse complementary strand encoding the last 15 amino acid residues Cys-44 to Asn-58 in the synthetic single-stranded form of INSULIN (GGG). With these two primers, a synthetic single-stranded form of INSULIN (GGG) was generated by annealing, extending the annealed primer, digesting with SalI and ClaI, and subcloning into pScCHSA digested with XhoI / ClaI. . The NotI fragment from this clone was then ligated to the NotI site of pSAC35 to generate construct ID 2278. Construct ID number 2278 encodes an albumin fusion protein comprising a chimeric leader sequence, a synthetic single chain of INSULIN (GGG), and a mature form of HSA.

  Further analysis of the N-terminus of the expressed albumin fusion protein by amino acid sequencing should confirm the presence of the expected INSULIN (GGG) sequence (see below).

The INSULIN albumin fusion protein of the invention is preferably a mature form of HSA (ie Asp) fused to either the N-terminus or C-terminus of a synthetic single-chain analog of INSULIN (ie Phe-1 to Asn-58). The sequence corresponding to the C peptide in the middle region of the proinsulin cDNA was replaced by a synthetic linker (“GG ³² GPGKR”) (SEQ ID NO: 2148). In one embodiment of the invention, the INSULIN albumin fusion protein of the invention further comprises a signal sequence directed to the starting fusion polypeptide in the secretory pathway of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed and the mature INSULIN albumin fusion protein is secreted directly into the culture medium. The INSULIN albumin fusion protein of the present invention comprises a heterologous signal sequence known in the art (MAF, INV, Ig, fibrin B, clusterin, insulin-like growth factor binding protein 4, HSA leader sequence variant (chimeric HSA / MAF leader sequence Including, but not limited to), or other heterologous signal sequences. In a preferred embodiment, the INSULIN albumin fusion protein of the invention comprises native INSULIN. In a further preferred embodiment, the INSULIN albumin fusion protein of the present invention further comprises an N-terminal methionine residue. Polynucleotides encoding these polypeptides (including fragments and / or variants) are also encompassed by the present invention.

(Expression and purification of construct ID 2278)
(Expression in yeast S. cerevisiae)
Construct 2278 is obtained from yeast S. cerevisiae by methods known in the art. can be transformed with C. cerevisiae (see Example 3). Expression levels can be tested by immunoblot detection using anti-HSA serum as the primary antibody.

(Purification from the supernatant of yeast S. cerevisiae cells)
Yeast S. In cerevisiae, the cell supernatant containing the secreted INSULIN (GGG) albumin fusion protein expressed from construct ID number 2278 can be purified as described in Example 4. The N-terminal sequencing of this expressed and purified albumin fusion protein was performed using FVNQH (SEQ ID NO: 2149) corresponding to the amino terminus of a long acting insulin analog (INSULIN (CG ³² G)) in a synthetic single chain form. Should occur.

In vitro [ ³ H] -2-deoxyglucose uptake assay in the presence of albumin fusion protein encoded by construct 2278
(Method)
An in vitro assay to measure glucose uptake in 3T3-L1 adipocytes in the presence of the INSULIN (GGG) albumin fusion protein encoded by construct 2278 can be performed as described below in Example 41. . Other assays known in the art that can be used to test INSULIN (GGG) albumin fusion proteins include L6 rat myoblast proliferation assay via glycogen synthesis kinase-3 (GSK-3) and rat malate enzyme H4IIe reporter assay including promoter (rMEP) -SEAP reporter, sterol regulatory element binding protein (SREBP) -SEAP reporter, fatty acid synthase (FAS) -SEAP reporter, and phosphoenolpyruvate carboxykinase (PEPCK) -SEAP reporter See Example 48), but is not limited thereto.

In vitro pancreatic cell line proliferation assay in the presence of albumin fusion protein encoded by construct 2278
(Method)
Measure the differentiation and proliferation of ductal epithelial pancreatic ARIP cell line into insulin producing β cells in the presence of INSULIN (GGG) albumin fusion protein encoded by construct 2278 and / or the growth of insulin producing RIN-Mβ cell line An in vitro assay to measure can be performed as described below under the heading "Example 42: In vitro assay of [ ³ H] thymidine incorporation into pancreatic cell lines".

(The activity of the albumin fusion protein encoded by construct 2278 can be assayed in vivo using a diabetic NOD and / or NIDDM mouse model)
The activity of the INSULIN (GGG) albumin fusion protein encoded by construct 2278 was determined by the titles “Example 44: Development of diabetes in NOD mice”, “Example 45: Histological examination of NOD mice”, and “Example 47”. Can be measured using the NOD and / or NIDDM mouse model described below under “: In vivo mouse model of NIDDM”.

(Example 37: Indicator for insulin albumin fusion protein)
The results from the in vitro assay described above show that insulin albumin fusion protein can treat and prevent hyperglycemia, insulin resistance, insulin deficiency, hyperlipidemia, hyperketonuria, and diabetes, type 1 diabetes and type 2 diabetes. And / or useful for diagnosis.

Example 38: Preparation of HSA-hGH fusion protein
HSA-hGH fusion protein was prepared as follows.

(Cloning of hGH cDNA)
hGH cDNA was obtained by PCR amplification from a human pituitary cDNA library (Cat. No. HL1097v, Clontech Laboratories, Inc.). Two oligonucleotides (HGH1 and HGH2) suitable for PCR amplification of hGH cDNA were synthesized using an Applied Biosystems 380B Oligonucleotide Synthesizer.

ＨＧＨ１およびＨＧＨ２は、ｈＧＨ ｃＤＮＡ配列（Ｍａｒｔｉａｌら，１９７９）の同じ位置のものとは、ＰＣＲ増幅後、ＥｃｏＲＩ部位をこのｃＤＮＡの５’末端に導入し、そしてＢａｍＨＩ部位をこのｃＤＮＡの３’末端に導入するように、それぞれ、２個および３個のヌクレオチドで異なった。さらに、ＨＧＨ２は、ｈＧＨ配列のすぐ下流にＨｉｎｄＩＩＩ部位を含んだ。

HGH1 and HGH2 are identical to those at the same position in the hGH cDNA sequence (Martial et al., 1979) after PCR amplification, introducing an EcoRI site at the 5 ′ end of the cDNA and a BamHI site at the 3 ′ end of the cDNA. As introduced, they differed by 2 and 3 nucleotides, respectively. In addition, HGH2 contained a HindIII site immediately downstream of the hGH sequence.

  PCR amplification using the Perkin-Elmer-Cetus Thermal Cycler 9600 and Perkin-Elmer-Cetus PCR kits was performed using a single stranded DNA template isolated from phage particles of a cDNA library as follows: 10 μL Of phage particles were lysed by the addition of 10 μL of phage lysis buffer (280 μg / mL proteinase K in TE buffer) and incubated at 55 ° C. for 15 minutes, followed by incubation at 85 ° C. for 15 minutes. After incubation on ice for 1 minute, phage debris was pelleted by centrifuging at 14,000 rpm for 3 minutes. The PCR mixture contained 6 μL of this DNA template, 0.1 μM of each primer and 200 μM of each deoxyribonucleotide. PCR is performed in 30 cycles of denaturation at 94 ° C. for 30 seconds, annealing at 65 ° C. for 30 seconds and extension at 72 ° C. for 30 seconds (increase 1 second extension time per cycle). .

  Analysis of this reaction by gel electrophoresis showed a single product of the expected size (589 base pairs).

  The PCR product was purified using the Wizard PCR Preps DNA Purification System (Promega Corp) and then digested with EcoRI and HindIII. After further purification of the EcoRI-HindIII fragment by gel electrophoresis, this product was cloned into pUC19 (GIBCO BRL) digested with EcoRI and HindIII to provide pHGH1. DNA sequencing of the EcoRI HindIII region confirmed that the PCR product is identical in sequence to the hGH sequence (Martial et al., 1979), except for the 5 'and 3' ends that introduced the EcoRI and BamHI sites, respectively. Indicated.

(Expression of hGH cDNA)
To form pBST (+), the polylinker sequence of the phagemid pBluescript (+) (Stratagene) was replaced by inserting an oligonucleotide linker, and two 75-mer oligonucleotides were inserted between EcoRI and HindIII. Formed by annealing. The new polylinker contained a unique NotI site.

  To form pHSA1, the NotI HSA expression cassette of pAYE309 (EP 431 880) containing the PRBI promoter, DNA encoding the HSA / MFα-1 hybrid leader sequence, DNA encoding HSA and the ADH1 terminator was expressed in pBST (+) I moved to. To form pHSA2, the HSA coding sequence was removed from this plasmid by digestion with HindIII followed by religation.

  The cloning of hGH cDNA provided an hGH coding region that lost the first 8 base pairs (bp) of the sequence of pro-hGH and mature hGH. To construct an expression plasmid for secretion of hGH from yeast, the yeast promoter, signal peptide and the first 8 bp of the hGH sequence were ligated to the 5 ′ end of the cloned hGH sequence as follows: two synthetic oligos Via nucleotides (HGH3 and HGH4), which can be annealed to each other in such a way as to produce a double-stranded fragment of DNA having a sticky end that can be annealed at the SfaNI sticky end and the EcoRI sticky end. The SfaNI fragment was ligated to the 5 ′ end of EcoRI / HindIII from pHGHI:

ｐＨＧＨ２を作るために、このように形成したＨｉｎｄＩＩＩフラグメントを、ＨｉｎｄＩＩＩで消化したｐＨＳＡ２にクローニングした。その結果、ｈＧＨ ｃＤＮＡをＰＲＢＩプロモーターおよびＨＳＡ／ＭＦα−１融合リーダー配列の下流に位置付けた（国際公開ＷＯ ９０／０１０６３を参照のこと）。ｐＨＧＨ１２を作るために、ｐＨＧＨ２において含まれるＮｏｔＩ発現カセット（ｈＧＨ ｃＤＮＡの下流にＡＤＨＩターミネーターを含んだ）を、ＮｏｔＩで消化したｐＳＡＣ３５（Ｓｌｅｅｐら、ＢｉｏＴｅｃｈｎｏｌｏｇｙ ８：４２（１９９０））にクローニングした。このプラスミドは、複製機能を提供するように２μｍプラスミド全体および形質転換株の選択のためのＬＥＵ２遺伝子を含んだ。

To make pHGH2, the HindIII fragment thus formed was cloned into pHSA2 digested with HindIII. As a result, hGH cDNA was positioned downstream of the PRBI promoter and HSA / MFα-1 fusion leader sequence (see International Publication WO 90/01063). To make pHGH12, the NotI expression cassette contained in pHGH2 (containing the ADHI terminator downstream of the hGH cDNA) was cloned into NotI digested pSAC35 (Sleep et al., BioTechnology 8:42 (1990)). This plasmid contained the entire 2 μm plasmid and the LEU2 gene for selection of transformants to provide replication function.

  pHGH12 was transformed into S. cerevisiae by transformation. cerevisiae D88 and individual transformants in 10 mL of YEPD (1% w / v yeast extract, 2% w / v, peptone, 2% w / v, glucose) at 30 ° C. for 3 days Allowed to grow.

  After centrifugation of the cells, the supernatant was examined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and of the expected size and anti-hGH antiserum (Sigma, Poole, UK) on Western blots It was found to contain a protein recognized by.

(Cloning and expression of HSA-hGH fusion protein)
In order to fuse the HSA cDNA to the 5 ′ end of the hGH cDNA, a fragment of pHSA1 HindIII-Bsu361 (which contains most of the HSA cDNA) is passed through two oligonucleotides (HGH7 and HGH8) to the pHGH1 EcoRI-HindIII fragment. (Including most of hGH cDNA).

ｐＨＧＨ１０を作るために、このように形成されたＨｉｎｄＩＩＩフラグメントを、ＨｉｎｄＩＩＩで消化したｐＨＳＡ２にクローニングし、そしてｐＨＧＨ１６を作るために、このプラスミドのＮｏｔＩ発現カセットを、ＮｏｔＩで消化したｐＳＡＣ３５にクローニングした。

To make pHGH10, the HindIII fragment thus formed was cloned into pHSA2 digested with HindIII, and the NotI expression cassette of this plasmid was cloned into NotI-digested pSAC35 to make pHGH16.

  S. pHGH16 was used to transform cerevisiae D88 and the culture supernatant was analyzed as described above. A dominant band with a molecular weight of approximately 88 kD was observed, which was consistent with the combined mass of HSA and hGH. Western blot using anti-HSA antiserum and anti-hGH antiserum (Sigma) confirmed the presence of two components of the albumin fusion protein.

  The albumin fusion protein was purified from the culture supernatant by cation exchange chromatography followed by anion exchange and gel permeation chromatography. N-terminal analysis of this protein by amino acid sequencing confirmed the presence of the expected albumin sequence.

An in vitro assay of growth hormone activity (Ealey et al., Growth Regulation 5:36 (1995)) showed that the albumin fusion protein retains complete hGH activity. In the rat pituitary excised rat weight gain model (performed essentially as described in the European Pharmacopoeia (1987, Book 556)), the same number of active units (based on the in vitro assay described above) When administered daily, this fusion molecule was more potent than hGH. Further experiments in which the albumin fusion protein was administered once every 4 days showed a global proliferative response similar to daily administration of hGH. Pharmacokinetic experiments in which ¹²⁵ I-labeled protein was administered to rats showed an approximately 10-fold increase in circulating half-life for albumin fusion protein compared to hGH.

  S. A similar plasmid that replaces the DNA encoding the cerevisiae invertase (SUC2) leader sequence with the sequence for the hybrid leader such that the encoded leader sequence and HSA are linked to the sequence (↓) as follows: It was constructed:

Ｓ．ｃｅｒｅｖｉｓｉａｅ ＤＢＩへの導入に関して、このプラスミドは、ｐＨＧＨ１６で観察されるものと類似のレベルで、アルブミン融合タンパク質の発現および分泌に指向した。このアルブミン融合タンパク質のＮ末端の分析は、成熟タンパク質からリーダー配列の正確で効率的な切断を示した。

S. For introduction into cerevisiae DBI, this plasmid was directed to the expression and secretion of albumin fusion protein at a level similar to that observed with pHGH16. Analysis of the N-terminus of this albumin fusion protein showed accurate and efficient cleavage of the leader sequence from the mature protein.

(Cloning and expression of hGH-HSA fusion protein)
To fuse the hGH cDNA to the 5 ′ end of the HSA cDNA, the HSA cDNA was first altered by site-directed mutagenesis to introduce an EcoN1 site near the 5 ′ end of the coding region. This was performed by the method of Kunkel et al. (Methods in Enzymol. 154: 367 (1987)) using a single-stranded DNA template prepared from pHSAI and a synthetic oligonucleotide (LEU4):

このオリゴヌクレオチドを使用する部位特異的変異誘発は、ＨＳＡ ｃＤＮＡのコード領域をＬｙｓ４からＬｅｕ４に変化させた（Ｋ４Ｌ）。しかし、２つのオリゴヌクレオチド（ＨＧＨ５およびＨＧＨ６）を介して、ｐＨＧＨ２ Ｎｏｔ１−ＢａｍＨＩフラグメントを突然変異させたｐＨＳＡＩのＥｃｏＮＩ−Ｎｏｔ１フラグメントに連結することによって、ｈＧＨ ｃＤＮＡを続いて５’末端に連結した場合、この変化を修復した：

Site-directed mutagenesis using this oligonucleotide changed the coding region of the HSA cDNA from Lys4 to Leu4 (K4L). However, when the hGH cDNA is subsequently ligated to the 5 ′ end by ligating the pHGH2 Not1-BamHI fragment to the mutated EcoNI-Not1 fragment via two oligonucleotides (HGH5 and HGH6), Repaired this change:

ｐＨＧＨ１４を作るために、このように形成したＮｏｔ１フラグメントを、Ｎｏｔ１で消化したｐＳＡＣ３５にクローニングした。Ｓ．ｃｅｒｅｖｉｓｉａｅ Ｄ８８を形質転換するためにｐＨＧＨ１４を使用し、そして培養上清を上記のように分析した。ｈＧＨおよびＨＳＡを組み合わせた質量に一致するおよそ８８ｋＤの分子量を有する優位なバンドが観察された。抗ＨＳＡ抗血清および抗ｈＧＨ抗血清を使用するウェスタンブロットは、このアルブミン融合タンパク質の２つの構成部分の存在を確認した。

To make pHGH14, the Not1 fragment thus formed was cloned into pSAC35 digested with Not1. S. pHGH14 was used to transform cerevisiae D88 and the culture supernatant was analyzed as described above. A dominant band with a molecular weight of approximately 88 kD consistent with the combined mass of hGH and HSA was observed. Western blot using anti-HSA antiserum and anti-hGH antiserum confirmed the presence of two components of this albumin fusion protein.

  The albumin fusion protein was purified from the culture supernatant by cation exchange chromatography followed by anion exchange and gel permeation chromatography. N-terminal analysis of this protein by amino acid sequencing confirmed the presence of the expected hGH sequence.

  In vitro studies have shown that this albumin fusion protein retains hGH activity but, as described above, is significantly less potent than an albumin fusion protein containing full length HSA (1-585) as the N-terminal position and hGH as the C-terminal position Showed that.

(Construction of plasmid for expression of fusion of hGH to HSA domain)
A fusion polypeptide was made in which the hGH molecule was fused to the first two domains of HSA (residues 1-387). Fusion to the N-terminus of hGH was accomplished by ligating the pHSA1 HindIII-Sap1 fragment containing most of the coding sequences for HSA domains 1 and 2 to the pHGHI EcoR1-HindIII fragment via oligonucleotides HGH11 and HGH12. .

ｐＨＧＨ３７を作るために、このように形成したＨｉｎｄＩＩＩフラグメントを、ＨｉｎｄＩＩＩで消化したｐＨＳＡ２にクローニングし、そしてこのプラスミドのＮｏｔ１発現カセットをＮｏｔ１で消化したｐＳＡＣ３５にクローニングした。

To make pHGH37, the thus formed HindIII fragment was cloned into HindIII digested pHSA2, and the Not1 expression cassette of this plasmid was cloned into Not1 digested pSAC35.

  The resulting plasmid (pHGH38) is S. cerevisiae. When transformed into cerevisiae DB1, an expression cassette was found that was found to direct secretion of the fusion polypeptide into the supernatant. Western blot using anti-HSA antiserum and anti-hGH antiserum confirmed the presence of two components of this albumin fusion protein.

  Albumin fusion protein was purified from the culture supernatant by cation exchange chromatography followed by gel permeation chromatography.

  In vivo studies with purified proteins have shown that circulation half-life is longer than that of hGH and, as mentioned above, albumin fusion proteins containing full length HSA (1-585) as the N-terminal position and hGH as the C-terminal position. It was shown that it was similar. In vitro studies have shown that this albumin fusion protein retains hGH activity.

  Using a similar strategy as detailed above, an albumin fusion protein containing the first domain of HSA (residues 1-194) as the N-terminal portion and hGH as the C-terminal portion was cloned and S . It was expressed in C. cerevisiae DBL. Western blotting of culture supernatants using anti-HSA and anti-hGH antisera confirmed the presence of two components of the albumin fusion protein.

(Fusion of HSA to hGH using flexible linker sequence)
A flexible linker (containing a repeating unit of [Gly-Gly-Gly-Gly-Ser) _n (SEQ ID NO: 2150), where n was either 2 or 3) was added to the oligonucleotide HGH16: , HGH17, HGH18 and HGH19 were introduced between HSA and hGH albumin fusion protein.

ＨＧＨ１６のＨＧＨ１７とのアニーリングが、ｎ＝２を生じ、その一方で、ＨＧＨ１９にアニールされたＨＧＨ１８は、ｎ＝３を生じた。アニーリング後、二本鎖オリゴヌクレオチドを、ｐＨＧＨ１から単離されたＥｃｏＲＩ−Ｂｓｕ３６１フラグメントを、Ｂｓｕ３６１消化ｐＨＧＨ１０にクローン化し、ｐＨＧＨ５６（ここで、ｎ＝２）およびｐＨＧＨ５７（ここで、ｎ＝３）を作製した。これらのプラスミドからのＮｏｔＩ発現カセットを、ＮｏｔＩ消化ｐＳＡＣ３５にクローン化し、それぞれ、ｐＨＧＨ５８およびｐＨＧＨ５９を作製した。

Annealing of HGH16 with HGH17 yielded n = 2, while HGH18 annealed to HGH19 yielded n = 3. After annealing, double-stranded oligonucleotides were cloned from EcoRI-Bsu361 fragment isolated from pHGH1 into Bsu361 digested pHGH10, creating pHGH56 (where n = 2) and pHGH57 (where n = 3) did. NotI expression cassettes from these plasmids were cloned into NotI digested pSAC35, creating pHGH58 and pHGH59, respectively.

  Cloning of the oligonucleotides to make pHGH56 and pHGH57 introduced a BamHI site into the linker sequence. Thus, ligating the HindIII-BamHI fragment from pHGH56 to the BamHI-HindIII fragment from pHGH57 (making n = 1), or linking the HindIII-BamHI fragment from pHGH57 to the BamHI-HindIII fragment from pHGH56. It was possible to construct a linker sequence where n = 1 and n = 4. Cloning of these fragments into the HindIII site of pHSA2 yielded pHGH60 (where n = 1) and pHGH61 (where n = 4). NotI expression cassettes from pHGH60 and pHGH61 were cloned into NotI digested pSAC35, creating pHGH62 and pHGH63, respectively.

  S. Transformation of cerevisiae with pHGH58, pHGH59, pHGH62, and pHGH63 resulted in transformants that secreted the fusion protein into the supernatant. Western blotting using anti-HSA and anti-hGH antisera confirmed the presence of two components of the albumin fusion protein.

  Albumin fusion protein was purified from the culture supernatant by cation exchange chromatography and then purified by anion exchange chromatography and gel permeation chromatography. Analysis of the N-terminus of the protein by amino acid sequencing confirmed the presence of the expected albumin sequence. Analysis of the purified protein by electrospray mass spectrometry shows that 315 D (n = 1), 630 D (n = 2), 945 D (n = 3) and 1260 D compared to the HSA-hGH fusion protein described above. An increase in mass of (n = 4) was confirmed. The purified protein was confirmed to be active in vitro.

  The hGH albumin fusion proteins of the present invention preferably comprise a mature form of HSA (ie, Asp-25 to Leu-609) fused to either the N-terminus or C-terminus of the mature form of hGH. In one embodiment of the invention, the hGH albumin fusion protein of the invention further comprises a signal sequence directed to the nascent fusion protein in the host secretory pathway used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed and the mature hGH albumin fusion protein is secreted directly into the culture medium. The hGH albumin fusion protein of the present invention may comprise a heterologous signal sequence, including MAF, INV, Ig, fibrin B, clusterin, insulin-like growth factor binding protein 4, a variant HSA leader sequence (chimeric HSA / MAF leader sequences, or other heterologous signal sequences known in the art, including but not limited to). In a preferred embodiment, the hGH albumin fusion protein of the present invention comprises native hGH. In a further preferred embodiment, the hGH albumin fusion protein of the present invention further comprises an N-terminal methionine residue. Polynucleotides encoding these polypeptides (including fragments and / or variants) are also encompassed by the present invention.

(Increasing shelf life of HSA-hGH fusion protein: method)
HSA-hGH and hGH were separately diluted in cell culture medium containing 5% horse serum to a final concentration of 100-200 μg / ml and incubated at 4 ° C., 37 ° C. or 50 ° C. At zero time and once a week thereafter, aliquots of the sample were tested for their biological activity in the Nb2 cell proliferation assay, and the data was biological activity of the control (hGH solution at time zero). Normalized for. In other assays, hGH and HSA-hGH were incubated at 4 ° C., 37 ° C., and 50 ° C. in phosphate buffer.

Nb2 cell proliferation assay: The proliferation of these cells depends on hGH or other mammary stimulating hormone. In a representative experiment, 10 ⁴ cells / well are placed in an incubator in 96 well plates in the presence of different concentrations of hGH or HSA-hGH in media containing 5-10% horse serum (eg, DMEM). Plate for 24-48 hours. After the incubation period, 1:10 volume of MTT (5 mg / ml in H ₂ O) is added to each well and the plate is incubated for an additional 6-16 hours. Proliferating cells convert MTT to insoluble formazan. Formazan is solubilized by acidic isopropanol and the color produced is measured at 570 nm on a microtiter plate reader. The degree of formazan formation reflects the level of cell proliferation.

(Increasing shelf life of HSA-hGH fusion protein: results)
Fusion of the therapeutic protein to albumin confers stability in aqueous or other solutions. The shelf life of the HSA fusion protein is extended with respect to the biological activity of HSA-hGH remaining after storage in cell culture medium for up to 5 weeks at 37 ° C. A solution of 200 μg / ml HSA-hGH is prepared in tissue culture medium containing 5% bovine serum and this solution starts at zero hours and is incubated at 37 ° C. At the indicated times, samples were removed and tested for their biological activity in the Nb2 cell assay at a final concentration of 2 ng / ml. The biological activity of HSA-hGH remains essentially intact (within experimental variations) after 5 weeks of incubation at 37 ° C. Recombinant hGH used as a control for this experiment lost its biological activity in the first week of the experiment.

  HSA-hGH was stable after storage of cell culture medium at 4 ° C, 37 ° C, or 50 ° C for up to 3 weeks. At time zero, a solution of HSA-hGH was prepared in tissue culture medium containing 5% bovine serum and incubated at 4 ° C, 37 ° C, or 50 ° C. At the indicated period, the sample was removed and assayed for its biological activity in the Nb2 cell assay at a final concentration of 60 ng / ml. HSA-hGH retains over 90% of its initial activity at all temperatures tested for at least 3 weeks after incubation, while hGH loses its biological activity within the first week. . This level of activity is further maintained at 37 ° C for at least 7 weeks and 50 ° C for at least 5 weeks. These results indicate that HSA-hGH is highly stable in aqueous solution even under temperature stress.

  The biological activity of HSA-hGH was stable compared to hGH in the Nb2 cell proliferation assay. Nb2 cells were grown in the presence of increasing concentrations of recombinant hGH, added at time zero. Cells were incubated for 24 or 48 hours, after which the extent of proliferation was measured by the MTT method. The increased stability of HSA-hGH in the assay results in essentially the same proliferative activity at 24 hours as 48 hours, while hGH shows a significant decrease in proliferative activity after 48 hours of incubation. . Compared to hGH, HSA-hGH has a lower biological efficacy after one day, and albumin fusion proteins are one-fifth more effective than hGH. However, after 2 days, HSA-hGH shows essentially the same efficacy as hGH due to the short lifetime of hGH in the assay. This increased stability of hGH as an albumin fusion protein has a major unexpected impact on the biological activity of the protein.

(Example 39: Instruction of hGH albumin fusion protein)
The results from in vitro and in vivo assays show that hGH albumin fusion protein is acromegaly, growth deficiency, growth failure and endogenous growth hormone replacement, growth hormone deficiency, growth deficiency and growth retardation Pradervilli Syndrome, growth defect, proliferation deficiency associated with chronic renal dysfunction, postmenopausal osteoporosis, burns, cachexia, cancer cachexia, dwarfism, metabolic disorders, obesity, renal failure, Turner syndrome (Pediatrics and adults), connective tissue inflammation, fracture treatment, weakness, or AIDS leprosy can be used to treat, prevent, detect, diagnose, and / or ameliorate.

Example 40: Isolation of selected cDNA clones from deposited samples
Many of the albumin fusion constructs of the present invention have been deposited with the ATCC as shown in Table 3. The albumin fusion construct may comprise any one of the following expression vectors: yeast S. cerevisiae. cerevisiae expression vector: pSAC35, mammalian expression vector pC4, or mammalian expression vector pEE12.1.

  pSAC35 vector (Sleep et al., 1990, Biotechnology 8:42), pC4 vector (ATCC accession number 209646; Cullen et al., Molecular and Cellular Biology, 438-447 (1985); Bosart et al., Cell 41: 521-530 (1985)). And pEE12.1 vector (Lonza Biologics, Inc .; Stephens and Cockett, Nucl. Acids Res. 17: 7110 (1989); International Publication No. WO 89/01036; Murphy et al., Biochem J. 227: 277-279 (1991) Bebbington et al., Bio / Technology 10: 169-175 (1992); US Patent No. 5,122,464; International Publication No. WO86 / 05807), the ampicillin resistance gene for bacterial cell growth. These vectors and / or albumin fusion constructs containing them can be obtained from E. coli using techniques described in the art such as Hanahan. E. coli strains (eg, Stratagene XL-1 Blue (Stratagene Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla, Calif., 92037)) and thia-to-B with 100 μg / mL ampicillin. Spread on plates and grow overnight at 37 ° C.

  The deposited material in the sample assigned the ATCC accession number quoted in Table 3 for any given albumin fusion construct may also include one or more additional albumin fusion constructs, each containing a different albumin fusion protein. Code. Thus, deposits that share the same ATCC accession number include at least the albumin fusion construct identified in the corresponding column of Table 3.

  Two approaches can be used to isolate specific albumin fusion constructs from the deposited samples of plasmid DNA cited in Table 3 for albumin fusion constructs.

(Method 1: Screening)
First, using methods known in the art, albumin fusion constructs are deposited using the plasmid probe corresponding to SEQ ID NO: X for each construct ID number in Table 1, deposited plasmid DNA. Can be isolated directly by screening a sample. For example, a specific polynucleotide having 30-40 nucleotides can be synthesized using an Applied Biosystems DNA synthesizer according to the reported sequence. Oligonucleotides can be labeled with ³² P-γ-ATP using, for example, T4 polynucleotide kinase and purified according to conventional methods. (Eg, Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring, NY (1982).) Albumin fusion constructs from a given ATCC deposit are known to those skilled in the art, as described above. Appropriate hosts using technology (eg, XL-1 Blue (Stratagene)) (eg, these are provided by the vector supplier or in the relevant publications or patents cited above. ). Transformants are plated on 1.5% agar plates (containing a suitable selection agent (eg, ampicillin)) to a density of about 150 transformants (colony) per plate. These plates can be obtained by conventional methods for bacterial colony screening (eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition (1989), Cold Spring Harbor Laboratory Press, pages 1.93 to 1.93). 104) or other techniques known to those skilled in the art, using Nylon membranes.

(Method 2: PCR)
Alternatively, the DNA encoding a given albumin fusion protein hybridizes to, for example, the deposited albumin fusion constructs 5 ′ and 3 ′ to the DNA encoding the given albumin fusion protein 17-20 By using two primers of nucleotides, it can be amplified from a sample of the deposited albumin fusion construct having SEQ ID NO: X. The polymerase chain reaction is performed under conventional conditions, eg, in a reaction mixture (25 μl) with 0.5 μg of the above cDNA template. Conventional reaction mixture, 1.5 to 5 mm MgCl _2, is 0.01% (w / v) gelatin, dATP each 20μM, dCTP, dGTP, dTTP, each primer and 0.25 units of Taq polymerase 25pmol . 35 cycles of PCR (94 ° C. 1 minute denaturation; 55 ° C. 1 minute annealing; 72 ° C. 1 minute extension) are performed using a Perkin-Elmer Cetus automated thermal cycler. The amplified product is analyzed by agarose gel electrophoresis and the band with the expected molecular weight is excised and purified. The PCR product is shown to be the sequence selected by subcloning and the DNA product is sequenced.

Several methods are available for the identification of the 5 ′ non-coding part or 3 ′ non-coding part of genes that may not be present in the deposited clone. These methods include filter probing, clone enrichment using specific probes, and protocols similar or identical to 5 ′ “RACE” and 3 ′ “RACE” protocols known in the art. It is not limited to these. For example, methods similar to 5 'RACE can be used to generate 5' ends lacking the desired full-length transcript.
(Frommont-Racine et al., Nucleic Acids Res., 21 (7): 1683-1684 (1993)).

  Briefly, a specific RNA oligonucleotide is ligated to the 5 'end of a population of RNA, possibly containing full-length gene RNA transcripts. A primer set comprising a primer specific for the ligated RNA oligonucleotide and a primer specific for the known sequence of the gene of interest is used to PCR amplify the 5 'portion of the desired full-length gene. This amplified product can then be sequenced and used to generate a full-length gene.

This above method starts with total RNA isolated from the desired source, but poly-A + RNA can be used. The RNA preparation can then be treated with phosphatase, if necessary, to remove the 5 ′ phosphate group on the degraded or damaged RNA. This RNA can interfere with subsequent RNA ligase steps. The phosphatase should then be inactivated and the RNA is treated with tobacco acid pyrophosphatase to remove the cap structure present at the 5 ′ end of the messenger RNA. This reaction leaves a 5 ′ phosphate group at the 5 ′ end of the cap cleaved RNA, which can be ligated to the RNA oligonucleotide using T4 RNA ligase.

  This modified RNA preparation is used as a template for first strand cDNA synthesis using gene specific oligonucleotides. First strand synthesis reaction for PCR amplification of the desired 5 'end using primers specific for the ligated RNA oligonucleotide and primers specific for the known sequence of the gene of interest Use as a template. The resulting product is then sequenced and analyzed to confirm that the 5 'end sequence belongs to a given gene.

Example 41: [< ³ > H] -2-deoxyglucose uptake assay
Fat, skeletal muscle, and liver are insulin sensitive tissues. Insulin can stimulate glucose uptake / transport into these tissues. In the case of fat and skeletal muscle, insulin initiates signaling that ultimately leads to the translocation of glucose transporter 4 molecule (GLUT4) from the differentiated intracellular compartment to the cell surface. Once on the cell surface, GLUT4 allows glucose uptake / transport.

([ ³ H] -2-deoxyglucose uptake)
Many fat-related and muscle-related cell lines can be tested for glucose uptake / transport activity in the absence or presence of any one or more therapeutic drug combinations listed for the treatment of diabetes. In particular, 3T3-L1 mouse fibroblasts and L6 mouse skeletal muscle cells can be differentiated into 3T3-L1 adipocytes and myotubes, respectively, as suitable in vitro models for [ ³ H] -2-deoxyglucose uptake assays (Urso et al., J Biol Chem, 274 (43): 30864-73 (1999); Wang et al., J Mol Endocrinol, 19 (3): 241-8 (1997); Haspel et al., J Membr Biol, 169 (1 ): 45-53 (1999); Tsakiridis et al., Endocrinology, 136 (10): 4315-22 (1995)). Briefly, 2 × 10 ⁵ cells / 100 μL of adipocytes or differentiated L6 cells were transferred to 96-well tissue culture (“TC” treated) in post-differentiation medium (ie, 50 μg / mL poly-L- Lysine-coated) plates and incubated overnight at 37 ° C. in 5% CO ₂ . Cells were washed once with serum-free low glucose DMEM medium and then listed for treatment of 100 μL / well of the same medium and 100 μL / well of buffer or diabetes in the absence or presence of 1 nM insulin. Any combination of two or more therapeutic drugs (eg, increasing concentrations of 1 nM, 10 nM, and 100 nM of the therapeutic agent of the invention (eg, the specific fusion disclosed as SEQ ID NO: Y and fragments and variants thereof)) And depleted at 37 ° C. for 16 hours. The plate is washed 3 times with 100 μL / well HEPES buffered saline. Insulin was incubated at 37 ° C. in HEPES buffered saline in the presence of 10 μM labeled [ ³ H] -2-deoxyglucose (Amersham, # TRK672) and 10 μM unlabeled 2-deoxyglucose (SIGMA, D-3179). Add 1 nM for 30 min. As a control, the same conditions are carried out except in the absence of insulin. A final concentration of 10 μM cytochalasin B (SIGMA, C6762) is added to separate wells at 100 μL / well to measure non-specific uptake. Cells are washed 3 times with HEPES buffered saline. Labeled, ie 10 μM [ ³ H] -2-deoxyglucose and unlabeled, ie 10 μM 2-deoxyglucose, were added for 10 minutes at room temperature. Cells are washed three times with cold phosphate buffered saline “PBS”. Cells are lysed upon addition of 150 μL / well of 0.2 N NaOH followed by incubation with shaking for 20 minutes at room temperature. The sample is then transferred to a scintillation vial and 5 mL of scintillation fluid is added to it. Vials are counted in a β-scintillation counter. Uptake in duplicate conditions (the difference is the absence or presence of insulin) is determined using the following equation: [(insulin counts per minute “cpm” −nonspecific cpm) / ( Insulin-free cpm-non-specific cpm)]. The average response is within the limits of about 5 and 3 times that of controls on adipocytes and myotubes, respectively.

(Cell differentiation)
The cells are allowed to become completely confluent in a T-75 cm ² flask. Remove media and replace with 25 mL pre-differentiation media for 48 hours. Cells are incubated at 37 ° C. with 5% CO ₂ , 85% humidity. After 48 hours, the pre-differentiation medium is removed and replaced with 25 mL of differentiation medium for 48 hours. Cells are again incubated at 37 ° C. with 5% CO ₂ , 85% humidity. After 48 hours, the medium is removed and replaced with 30 mL of post-differentiation medium. Post-differentiation medium is maintained for 14-20 days or until full differentiation is achieved. The medium is changed every 2-3 days. Human adipocytes can be purchased from Zen-Bio, INC (# SA-1096).

Example 42: In vitro assay of [ ³ H] -thymidine incorporation into pancreatic cell lines GLP-1 reduces differentiation of the rat pancreatic ductal epithelial cell line ARIP in a time-dependent and dose-dependent manner It was recently shown. This differentiation is associated with increased Islet Dual Homebox-1 (IDX-1) and insulin mRNA levels (Hui et al., 2001, Diabetes, 50 (4): 785-96). IDX-1 then increases mRNA levels of the GLP-1 receptor.

(Cell type tested)
(RIN-M cells)
These cells are available from the American Type Tissue Culture Collection (ATCC cell line number CRL-2057). The RIN-M cell line was derived from a radiation-induced transplantable rat islet cell tumor. This strain is established from tumor nude mouse xenografts. The cells produce and secrete islet polypeptide hormones and produce L-dopa decarboxylase, a marker for cells with amine precursor uptake and decarboxylation (ie, APUD) activity.

(ARIP cells)
There are exocrine pancreatic cells of epithelial morphology available from the American Type Tissue Culture Collection (ATCC cell line number CRL-1674). References: Jessop, N .; W. And Hay, R .; J. et al. Cocker, M., "Characteristics of two pancreatic exoline cell lines, derived from transplantable tumours," Vitro 16: 212, (1980); Et al., “Identification of a cell-specific DNA-binding activity that interacts with a transcriptional activator of genes expressed in the acinar mandala. Cell. Biol. 9: 2464-2476, (1989); Roux, E .; Et al., "The cell-specific transcription factor PTF1 contains two differential subunits that interact with the DNA" Genes Dev. 3: 1613-1624 (1989); and Hui, H .; Et al., "Glucagon-like peptide 1 inducements differentiation of isodile homeobox-1-positive pancreatic double cells into insulin-cells 7".

(Preparation of cells)
The RIN-M cell line was grown in RPMI 1640 medium (Hyclone, # SH300027.01) containing 10% fetal calf serum (Hyclone, # SH30088.03), at a ratio of 1: 3 to 1: 6. Planted every 8 days. The medium is changed every 3-4 days.

  ARIP (ATCC # CRL-1674) cell line was prepared from Ham's F12K medium (ATCC # 30-) containing 2 mM L-glutamine prepared to contain 1.5 g / L sodium bicarbonate and 10% fetal calf serum. In 2004). ARIP cell lines are subcultured twice a week at a ratio of 1: 3 to 1: 6. The medium is changed every 3-4 days.

(Assay protocol)
Cells were seeded at 4000 cells / well in 96 well plates and cultured for 48-72 hours to 50% association. Cells were changed to medium without serum at 100 μL / well. After incubation for 48-72 hours, the serum and / or treatment of the test invention (eg, albumin fusion proteins and fragments of the invention and variants thereof) are added to the wells. Incubation continues for an additional 36 hours. [ ³ H] -Thymidine (5-20 Ci / mmol) (Amersham Pharmacia, # TRK120) is diluted to micro Curies / 5 microliters. After 36 hours of incubation, 5 microliters are added per well for an additional 24 hours. The reaction was terminated by gradual washing of the cells once with cold Phosphate-Buffered Saline, “PBS”. The cells are then fixed with 100 microliters of 10% ice cold TCA for 15 minutes at 4 ° C. PBS is removed and 200 microliters 0.2N NaOH is added. The plate is incubated for 1 hour at room temperature with agitation. The solution is transferred to a scintillation vial and 5 mL of scintillation fluid compatible with the aqueous solution is added and stirred vigorously. Vials are counted with a β scintillation counter. For negative controls, use buffer only. For positive controls, use fetal calf serum.

Example 43: Assay for Glycoline
Diabetes (ie, excess sugar in the urine) can be easily assayed to provide an indication of the disease state of diabetes. Excess urine in the patient sample when compared to normal patient samples is a symptom of IDDM and NIDDM. The effectiveness of treatment of patients suffering from IDDM and NIDDM is indicated by the resulting reduction in excess urine glucose levels. In preferred embodiments for monitoring IDDM and NIDDM, urine samples from patients are assayed for the presence of glucose using techniques known in the art. Diabetes in humans is defined by urinary glucose concentrations greater than 100 mg per 100 ml. Excess sugar levels in patients with diabetes can be measured more accurately by obtaining a blood sample and assaying serum glucose.

(Example 44: Development of diabetes in NOD mice)
Although female NOD (non-obese diabetic) mice are characterized by representing IDDM using a course similar to that found in humans, this disease is more severe in female NOD mice than in male NOD mice. Hereinafter, unless otherwise specified, the term “NOD mouse” refers to a female NOD mouse. NOD mice have a progressive decay of beta cells caused by chronic autoimmune diseases. Thus, NOD mice begin to live with euglycemia or normal blood glucose levels. However, by about 15-16 weeks of age, NOD mice begin to have high blood glucose levels. This indicates that most of the pancreatic beta cells of the mouse have collapsed and correspondingly the pancreas is unable to produce enough insulin. Thus, both the cause and progression of the disease is similar to human IDDM patients.

  In vivo assays for the effectiveness of the immunization regimen can be evaluated in female NOD / LtJ mice (commercially available from The Jackson Laboratory, Bar Harbor, Me). It has been reported in the literature that 80% of female mice develop diabetes by 24 weeks of age and the development of insulitis progresses between 6 and 8 weeks of age. NOD mice are inbred and are highly responsive to various immunoregulatory strategies. Adult NOD mice (6-8 weeks old) have an average weight of 20-25 g.

  These mice may be untreated (control) or alone or in combination with the other therapeutic agent compositions described above (eg, the albumin fusion proteins of the invention, as well as their Fragments and variants). The effectiveness of these various treatments on the progression of diabetes can be measured as follows.

  At 14 weeks of age, female NOD mice can be phenotyped according to glucose tolerance. Glucose tolerance can be measured with the intraperitoneal glucose tolerance test (IPGTT). Briefly, blood is drawn from the paraorbital plexus at 0 minutes and 60 minutes after glucose intraperitoneal injection (1 g / kg body weight). Normal tolerance is defined as less than 144 mg% plasma glucose at 0 minutes or less than 160 mg plasma glucose at 60 minutes. Blood glucose levels can be determined using a Glucometer Elite device.

  Based on this phenotypic analysis, animals can be allocated to different experimental groups. Specifically, animals with higher blood glucose levels can be assigned to an incapable glucose tolerance group. These mice can optionally be fed and supplied with acidified water (pH 2.3).

  Glucose-tolerant and glucose intolerant mice can be further divided into control groups, albumin fusion protein groups of the invention, and albumin fusion protein / therapeutic agent composition combination groups. Control group mice can receive daily (6 times per week) intraperitoneal injection of vehicle. Mice of the albumin fusion protein group can receive intraperitoneal injections of the therapeutic agents of the invention (eg, the albumin fusion proteins of the invention and fragments and variants thereof) in vehicle daily (six times per week). Mice of the albumin fusion protein / therapeutic agent composition combination group can receive both the albumin fusion protein and the combination of therapeutic agents described above.

  Urinary glucose levels in NOD mice can be determined on a weekly basis using Labstix (Bayer Diagnostics, Hampshire, England). Body weight and fluid intake can also be determined on a weekly basis. The onset of diabetes is defined after the onset of diabetes for two consecutive decisions. Ten weeks after treatment, further IPGTT can be performed and the animals can be sacrificed the next day.

  Over a 10 week course of treatment, control animals in both the glucose tolerant and glucose intolerant groups develop diabetes at a rate of 60% and 86%, respectively (Gross et al., US Pat. No. 5,866,546). checking). Thus, diabetes occurs at a high rate even in NOD mice that are initially glucose tolerant when no treatment intervention is made.

  Results can be confirmed by measuring blood glucose levels in NOD mice before and after treatment. In all described groups, blood glucose levels in both glucose tolerant and glucose intolerant mice are measured as described above.

  In an alternative embodiment, the therapeutic agents of the invention (eg, the specific fusion proteins disclosed in SEQ ID NO: Y and fragments and variants thereof) can be quantified using spectroscopic analysis and prior to injection, Appropriate amount of protein is 50.50 per dose. mu. may be resuspended in 1 phosphate buffered saline (PBS). Two injections every other week can be administered subcutaneously under the dorsal skin of each mouse. Before immunization, monitoring can be performed on two separate occasions, and monitoring can be performed weekly throughout the treatment, and can continue thereafter. Urine can be tested weekly for glucose levels (Keto-Diastix.RTM .; Miles Inc., Kankake, Ill.) And diabetic mice can be checked for serum glucose (MediSense, Inc. Waltham, Mass). Diabetes can be analyzed when blood sugar levels at starvation are greater than 2.5 g / L.

(Example 45: Histological examination of NOD mice)
Histological examination of tissue samples from NOD mice shows that the combination of the composition of the invention and / or the composition of the invention and other therapeutic agents for diabetes increases the relative concentration of β cells in the pancreas The ability to do so can be demonstrated. The experimental method is as follows.

  Mice from Example 44 can be sacrificed at the end of the treatment period and tissue samples from the pancreas can be removed. These samples can be fixed in 10% formalin in 0.9% saline and embedded in wax. For immunolabeling, five series of 5. mu. Two sets of m intercepts, 150. mu. Can be cut at m cutting intervals. Sections can be immunolabeled for insulin (guinea pig anti-insulin antiserum, 1: 1000 dilution, ICN Themes UK) and glucagon (rabbit anti-glucagon antiserum, 1: 2000 dilution) and peroxidase-conjugated anti-hamster Antiserum (Dako, High Wycombe, UK) or peroxidase-conjugated anti-rabbit antiserum (1:50 dilution, Dako) can be used for detection.

  The composition of the present invention has an effect on the visual weight of beta cells that is as strong as it does for clinical symptoms of diabetes in glucose tolerant or glucose intolerant animals. It does not have to be.

(Example 46: Pancreatic β-cell transplantation combination treatment)
Transplantation is a common form of treatment of autoimmune diseases, especially when the target self tissue is severely damaged. For example, pancreas transplantation and Langerhans islet cell transplantation are common treatment options for IDDM, but are not limited to these (eg, Stewatt et al., Journal of Clinical Endocrinology & Metabolism 86 (3) 984-988 (2001); Brunicali, Transplant.Proc.28: 2138-40 (1996); Kendall & Robertson, Diabats Metab.22: 157-163 (1996); Hamano et al., Kobe J. Med.Sci.42: 93-104 (1996); Stratta, Diabats Metab.22: 139-146 (1996); and Kikhabwala et al., Am.J. Su. rg.171: 516-520 (1996)). With respect to any transplantation method, transplantation therapy for patients with autoimmune disease includes treatment to minimize the risk of rejection of the transplanted tissue from the host. However, autoimmune diseases involve an additional distinct risk that pre-existing host autoimmune responses that have damaged the original self tissue will have the same damaging effect on the transplanted tissue. Accordingly, the present invention provides a method for the treatment of autoimmune pancreatic disease using an albumin fusion protein of the present invention in combination with an immunomodulator and / or an immunosuppressant in an individual receiving autoimmune disease transplant therapy. And compositions.

  In accordance with the present invention, the albumin fusion-based compositions and formulations described above are used to treat damage to transplanted organs, transplanted tissues, or transplanted cells that result from the host individual's autoimmune response initially directed against the original self-tissue. Administer to prevent or treat. Administration can be performed every other week at 2-4 doses both before and after transplantation.

  In Langerhans islet cell transplantation or pancreas transplantation, the following immunomodulators and / or immunosuppressants may be used with the albumin fusion therapeutics of the present invention: AI-401, CDP-571 (anti-TNF monoclonal antibody) CG-1088, Diamyd (diabetic vaccine), ICM3 (anti-ICAM-3 monoclonal antibody), linomid (Roquinimex), NBI-6024 (modified peptide ligand), TM-27, VX-740 (HMR-3480) , Caspase-8 protease inhibitor, thalidomide, hOKT3γ1 (Ala-ala) (anti-CD3 monoclonal antibody), oral interferon α, oral lactic acid bacteria, and LymphoStat-B®, but are not limited to these. .

Example 47: In vivo mouse model of NIDDM
Three-week-old male C57BL / 6J mice can be obtained from Jackson Laboratory (Bar Harbor, ME) and can be obtained from conventional diets or high fat diets (35.5 wt% by weight; Bioserv. Frenchtown, NJ) or high Either a fructose diet (60% by weight; Harlan Teklad, Madison, WI) may be fed. A regular diet consists of 4.5% fat by weight, 23% protein by weight, 31.9% carbohydrate by weight, 3.7% fructose by weight and 5.3% by weight. % Fiber. A high fat (Lard) diet is 35.5% fat by weight, 20% protein by weight, 36.4% carbohydrate by weight, 0.0% fructose by weight and 0.00% by weight. Consists of 1 wt% fiber. A high fructose diet consists of 5% fat by weight, 20% protein by weight, 0.0% carbohydrate by weight, 60% fructose by weight and 9.4% fiber by weight . No more than 5 animals per cage, housed in a room temperature controlled to 22 ° C. + / − 3 ° C. and 20% wet controlled, with 12 hours of sunshine (6 am-6pm) / dark cycle. (Luo et al., 1998, Metabolism 47 (6): 663-8, “Nongeneous mouse models of non-insulin-dependent diabates melitus”; of the long-acting GLP-1 derivative NN2211 inductors lasting and reversible weight loss in both normal and o ese rats "). Either streptozocin “STZ” (Sigma, St. Louis, MO) or vehicle (0.05 mol / L citric acid, pH 4.5) at 100 mg / kg body weight after exposure to each diet for 3 weeks Can be injected intraperitoneally into mice and maintained on the same diet for the next 4 weeks. Under non-starvation conditions, blood is obtained by cutting the distal portion of the tail 1 week, 2 weeks and 4 weeks after STZ. Samples are used to measure non-starved plasma glucose and insulin concentrations. Body weight and food intake are recorded weekly.

  To directly determine the effect of a high fat diet on the ability of insulin to stimulate glucose treatment, three groups of mice (fat diet fed mice, normal diet fed mice injected with vehicle, and the end of the 7 week period above) These experiments can be started on fat-fed mice injected with STZ. Prior to the experiment, the mice can be starved for 4 hours. In the first series of experiments, mice can be anesthetized with methylflurane (Pitman-Moor, Mundelein, IL) aspiration. Regular insulin (Sigma) can be injected intravenously ([IV], 0.1 U / kg body weight) via the tail vein and 3 minutes, 6 minutes, 9 minutes, 12 minutes and 15 minutes after injection. Blood can be collected from different tail veins after a minute. Plasma glucose concentrations can be determined for these samples and the half-life (t1 / 2) of glucose elimination from plasma using the pharmacokinetic / pharmacokinetic software program WinNonlin (Scientific Consulting, Apex, NC) Can be determined.

  In a second series of experiments, mice can be anesthetized with pentobarbital sodium (Sigma) in the peritoneum. The abdominal cavity was opened and the abdominal vein was exposed and catheterized with a 24 gauge IV catheter (Johnson-Johnson Medical, Arlington, TX). The catheter is secured to the muscle tissue adjacent to the abdominal vein, cut under the syringe connection, and hooked to a pre-filled PE50 plastic tube which is then connected to a syringe with infusion solution. The abdominal cavity is then sutured closed. With this approach, there is no blockage of blood backflow from the lower part of the fuselage. Mice were continuously infused with glucose (24.1 mg / kg / min) and insulin (10 mU / kg / min) at an injection volume of 10 μL / min. For measurement of plasma glucose concentration and plasma insulin concentration, retro-orbital blood samples (70 μL each) may be removed 90 minutes, 105 minutes, 120 minutes and 135 minutes after the start of infusion. The average of these four samples can be used to assess steady state plasma glucose concentration (SSPG) and steady state plasma insulin concentration (SSPI) for each animal.

  Finally, the albumin fusion protein, the therapeutic composition of the present application, converts plasma glucose either alone or in combination with one or more of the therapeutic drugs listed for the treatment of diabetes. Experiments to assess the ability to reduce are performed in the following two groups of “NIDDM” mouse models injected with STZ: (1) C57BL / 6J fed with fat diet, and (2) C57BL / 6J fed with fructose. . The mouse plasma glucose concentration for these studies can range from 255 to 555 mg / dL. Treatment of mice with the albumin fusion protein of the invention, either with vehicle or alone or in combination with any one or more of the therapeutic drugs listed for the treatment of diabetes Randomly assign to any of these. A total of three doses can be administered. Tail vein blood samples can be taken for measurement of plasma glucose concentration before the first dose and 3 hours after the last dose.

  An enzyme colorimetric assay, Glucose Diagnostic Kit from Sigma (Sigma # 315), can be used to determine plasma glucose concentration. Rat Insulin RIA Kit (# RI-13K; St. Charles, MO) from Linco Research can be used to determine plasma insulin levels.

Example 48 In Vitro H4IIe-SEAP Reporter Assay Establishing Involvement of Insulin Action
(Various H4IIe reporters)
H4IIe / rMEP-SEAP: Malate enzyme promoter (rMEP) isolated from rats contains a PPAR-γ element present in the insulin pathway. This reporter construct is stably transfected into the liver H4IIe cell line.

H4IIe / SREBP-SEAP: Sterol regulatory element binding protein (SREBP-1c) acts on the promoters of many insulin responsive genes (eg, fatty acid synthase (FAS)) and fibroblasts, brown adipocytes And a transcription factor that regulates the expression of genes important in fatty acid metabolism in hepatocytes. SREBP-1c is also known as brown adipocyte determinant differentiation factor 1 (ADD-1) and is regarded as the main mediator of the effect of insulin on gene expression in brown adipocytes.
Its activity is regulated by insulin levels, sterol levels and glucose levels. This reporter construct is stably transfected into the liver H4IIe cell line.

  The H4IIe / FAS-SEAP: fatty acid synthase construct contains a minimal SREBP-responsive FAS promoter. This reporter construct is stably transfected into the liver H4IIe cell line.

  The H4IIe / PEPCK-SEAP: phosphoenolpyruvate carboxykinase (PEPCK) promoter is a major site of hormonal control of PEPCK gene transcription that regulates PEPCK activity. PEPCK catalyzes an integrated and rate controlling step in liver gluconeogenesis and therefore must be carefully controlled to maintain blood glucose levels within normal limits. This reporter construct is stably transfected into the liver H4IIe cell line.

  This reporter construct can also be stably transfected into 3T3-L1 fibroblasts and L6 myoblasts. These stable cell lines are then differentiated into 3T3-L1 brown adipocytes and L6 myotubes as previously described in Example 41. These differentiated cell lines can then be used in the SEAP assay described below.

(Growth medium and assay medium)
Growth medium was 10% fetal bovine serum (FBS), 10% bovine serum, 1% NEAA, 1 × penicillin / streptomycin, and 0.75 mg / mL G418 (against H4IIe / rFAS-SEAP and H4IIe / SREBP-SEAP ) Or 0.50 mg / mL G418 (for H4IIe / rMEP-SEAP). For H4IIe / PEPCK-SEAP, the growth medium consists of 10% FBS, 1% penicillin / streptomycin, 15 mM HEPES buffered saline and 0.50 mg / mL G418.

  The assay medium for H4IIe / rFAS-SEAP reporter, H4IIe / SREBP-SEAP reporter and H4IIe / rMEP-SEAP reporter consists of low glucose DMEM medium (Life Technologies), 1% NEAA, 1 × penicillin / streptomycin. The assay medium for the H4IIe / PEPCK-SEAP reporter consists of 0.1% FBS, 1% penicillin / streptomycin and 15 mM HEPES buffered saline.

(Method)
96-well plates are seeded at 75,000 cells per well in 100 μL per well of growth medium until cells in logarithmic growth phase adhere. Cells are starved for 48 hours by replacing the growth medium with assay medium (200 μL per well). (For example, for H4IIe / PEPCK-SEAP, assay medium containing 0.5 μM dexamethasone is added at 100 μL per well and incubated for about 20 hours). This assay medium is then replaced with 100 μL fresh assay medium per well and obtained from a transfected cell line expressing the therapeutic agent of the invention (eg, the albumin fusion protein of the invention and fragments and variants thereof). A 50 μL aliquot of the collected cell supernatant is added to the wells. A cell line transfected with an empty vector is used as a negative control. Addition of 10 nM and / or 100 nM insulin to the well is used as a positive control. After 48 hours of incubation, conditioned medium is collected and SEAP activity is measured (Phospha-LightSystem protocol, Tropix # BP2500). Briefly, samples are diluted with dilution buffer and incubated at 65 ° C. for 30 minutes to inactivate endogenous non-placental forms of SEAP. A 50 μL aliquot of diluted sample is mixed with 50 μL of SEAP Assay Buffer containing an inhibitor mixture active against non-placental SEAP isozymes and incubated for an additional 5 minutes. A 50 μL aliquot of CSPD chemiluminescent substrate (which is diluted 1:20 in Emerald luminescence enhancer) is added to the mixture and incubated for 15-20 minutes. Plates are read with a Dynax plate luminometer.

Example 49: Preparation of HA-cytokine fusion protein or HA-growth factor fusion protein (eg, EPO, GMCSF, GCSF)
Various, including cDNA of the cytokine or growth factor of interest (eg, EPO) from a cDNA library, by RT-PCR, and by PCR using a series of overlapping synthetic oligonucleotide primers (all using standard methods) Can be isolated by these means. Nucleotide sequences for all these proteins are known and available (eg, US Pat. Nos. 4,703,088, 4,810,643 and 5,908,763). Prepare this cDNA at the 5 'and 3' ends to form restriction sites (restriction sites such as those where oligonucleotide linkers can be used) for cloning the cDNA into a vector containing the cDNA for HA Can do. This may or may not use a spacer sequence at the N-terminus or C-terminus. The EPO (or other cytokine) cDNA is cloned into a vector (eg, pPPC0005 (FIG. 2), pScCHSA, pScNHSA, or pC4): The HSA in which the complete expression cassette is present is then excised and inserted into the plasmid pSAC35. Thus allowing expression of the albumin fusion protein in yeast. Albumin fusion protein secreted from yeast can be collected and purified from the culture medium and tested for its biological activity. For expression in mammalian cell lines, a similar procedure applies, except that the expression cassette used uses a mammalian promoter, leader sequence and terminator (see Example 1). The expression cassette is then excised and inserted into a plasmid suitable for mammalian cell line transfection.

Example 50: Preparation of HA-IFN fusion protein (eg, IFNα)
The interferon cDNA of interest, such as IFNα, can be isolated by various means. Various means include, but are not limited to: RT-PCR or PCR using a series of overlapping synthetic oligonucleotide primers from a cDNA library, all using standard methods. The nucleic acid sequence of interferon (eg, IFNα) is known, for example, in US Pat. Nos. 5,326,859 and 4,588,585, and in EP 32,134, and in public databases such as GenBank. And available. The cDNA can be modified at the 5 ′ and 3 ′ ends to generate restriction sites. As a result, the oligonucleotide linker can be used to clone the cDNA into a vector for cloning the HA cDNA. This can be done with or without a spacer sequence at the N-terminus or C-terminus of the HA sequence. IFNα (or other interferon) cDNA is cloned into a vector such as pPPC0005 (FIG. 2), pScCHSA, pScNHSA, or pC4: HSA. The complete expression cassette is then excised from such a vector and inserted into the plasmid pSAC35 to allow expression of the albumin fusion protein in yeast. The albumin fusion protein secreted from the yeast can then be collected from the medium, purified and tested for its biological activity. A similar procedure applies for expression in mammalian cell lines, except that the expression cassette used uses a mammalian promoter, leader sequence and terminator (see Example 1). The expression cassette is then excised and inserted into a plasmid suitable for mammalian cell line transfection.

(Maximum protein recovery from vials)
The albumin fusion protein of the present invention is highly stable even when packaged at a low concentration. Moreover, despite the low protein concentration, good fusion protein recovery is observed even when the aqueous solution does not contain other proteins to minimize binding to the vial wall. The recovery of the HA-IFN solution contained in the vial was compared to the stocked solution. 6 or 30 [mu] g / ml HA-IFN solution was placed in a vial and stored at 4 [deg.] C. After 48 to 72 hours, the volume corresponding to the first 10 ng sample was removed and measured by IFN sandwich ELISA. The estimated volume was compared to the volume of the high concentration stock solution. As shown, in these vials, the sample was not substantially lost. This indicates that no extraneous material (eg, albumin) needs to be added to prevent sample loss to the vial wall.

(Stability and bioavailability of HA-α-IFN fusions in vivo)
To determine the stability and bioavailability of HA-α-IFN fusion molecules in vivo, purified fusion molecules (from yeast) were administered to monkeys. Pharmaceutical compositions formulated from HA-α-IFN fusions can account for prolonged serum half-life and bioavailability. Accordingly, the pharmaceutical composition may be formulated to include a lower dosage of α-interferon activity compared to the native α-interferon molecule.

  A pharmaceutical composition comprising an HA-α-IFN fusion can be used to treat or prevent a disease in a patient having any disease or disease state that can be modulated by administration of α-IFN. Such diseases include hairy cell leukemia, Kaposi's sarcoma, genital and anal warts, chronic hepatitis B, chronic non-A non-B hepatitis (especially hepatitis C), hepatitis D, chronic myelogenous leukemia, kidney Cell carcinoma, bladder carcinoma, ovarian carcinoma and cervical carcinoma, skin cancer, recurrent respiratory papillomatosis, non-Hodgkin T-cell lymphoma and cutaneous T-cell lymphoma, melanoma, multiple myeloma, AIDS, multiple sclerosis, glia But not limited to (see AHFS Drug Information, 1997, Interferon α).

  Accordingly, the present invention includes a HA-α-IFN fusion protein, polypeptide or peptide formulation comprising an appropriate dosage for human administration. The invention also includes a method of treating a patient in need of such treatment. Such methods include at least the step of administering a pharmaceutical composition comprising at least one HA-α-IFN fusion protein, polypeptide, peptide.

(Bifunctional HA-α-IFN fusion)
The HA-α-IFN expression vector may be modified to include an insert for expression of the bifunctional HA-α-IFN fusion protein. For example, the cDNA of the second protein of interest may be inserted in frame, downstream of the “rHA-α-IFN” sequence after removing the double stop codon or moving downstream of the coding sequence. Can do.

  In one variation of a bifunctional HA-α-IFN fusion protein, an antibody or fragment (GenBank accession number 4455139), or polypeptide, against a B lymphocyte stimulating protein is fused to one end of the HA component of the fusion molecule. obtain. This bifunctional protein is useful for modulating any immune response generated by the α-IFN component of the fusion.

Example 51: Preparation of HA-hormone fusion protein (eg, insulin, LH, FSH)
The cDNA of the hormone of interest (eg, insulin) can be isolated by various means. Various means include, but are not limited to: RT-PCR or PCR using a series of overlapping synthetic oligonucleotide primers from a cDNA library, all using standard methods. The nucleotide sequences of all these proteins are known and available in public databases such as GenBank. The cDNA can be modified at the 5 ′ and 3 ′ ends to generate restriction sites. As a result, the oligonucleotide linker can be used to clone the cDNA into a vector for cloning the HA cDNA. This can be done with or without a spacer sequence at the N-terminus or C-terminus. Hormonal cDNA is cloned into vectors such as pPPC0005 (FIG. 2), pScCHSA, pScNHSA, or pC4: HSA. The complete expression cassette is then excised from such a vector and inserted into the plasmid pSAC35 to allow expression of the albumin fusion protein in yeast. The albumin fusion protein secreted from the yeast can then be collected from the medium, purified and tested for its biological activity. A similar procedure applies for expression in mammalian cell lines, except that the expression cassette used uses a mammalian promoter, leader sequence and terminator (see Example 1). The expression cassette is then excised and inserted into a plasmid suitable for mammalian cell line transfection.
Example 52: Preparation of HA soluble receptor protein or HA binding protein fusion protein (eg, HA-TNF receptor)
The cDNA of the soluble receptor protein of interest, such as the TNF receptor, or binding protein can be isolated by various means. Various means include, but are not limited to: RT-PCR or PCR using a series of overlapping synthetic oligonucleotide primers from a cDNA library, all using standard methods. The nucleotide sequences of all these proteins are known and available in public databases such as GenBank. The cDNA can be modified at the 5 ′ and 3 ′ ends to generate restriction sites. As a result, the oligonucleotide linker can be used to clone the cDNA into a vector for cloning the HA cDNA. This can be done with or without a spacer sequence at the N-terminus or C-terminus. The receptor cDNA is cloned into a vector such as pPPC0005 (FIG. 2), pScCHSA, pScNHSA, or pC4: HSA. The complete expression cassette is then excised from such a vector and inserted into the plasmid pSAC35 to allow expression of the albumin fusion protein in yeast. The albumin fusion protein secreted from the yeast can then be collected from the medium, purified and tested for its biological activity. A similar procedure applies for expression in mammalian cell lines, except that the expression cassette used uses a mammalian promoter, leader sequence and terminator (see Example 1). The expression cassette is then excised and inserted into a plasmid suitable for mammalian cell line transfection.
Example 53: Preparation of HA-growth factor (eg, HA-IGF-1 fusion protein)
The cDNA of the growth factor of interest such as IGF-1 can be isolated by various means. Various means include, but are not limited to: RT-PCR or PCR using a series of overlapping synthetic oligonucleotide primers, all using standard methods (GenBank Accession). No. NP_000609). The cDNA can be modified at the 5 ′ and 3 ′ ends to generate restriction sites. As a result, the oligonucleotide linker can be used to clone the cDNA into a vector for cloning the HA cDNA. This can be done with or without a spacer sequence at the N-terminus or C-terminus. Growth factor cDNA is cloned into a vector such as pPPC0005 (FIG. 2), pScCHSA, pScNHSA, or pC4: HSA. The complete expression cassette is then excised from such a vector and inserted into the plasmid pSAC35 to allow expression of the albumin fusion protein in yeast. The albumin fusion protein secreted from the yeast can then be collected from the medium, purified and tested for its biological activity. A similar procedure applies for expression in mammalian cell lines, except that the expression cassette used uses a mammalian promoter, leader sequence and terminator (see Example 1). The expression cassette is then excised and inserted into a plasmid suitable for mammalian cell line transfection.
(Example 54: Preparation of HA-single chain antibody fusion protein)
Single chain antibodies are produced by several methods. Such methods include, but are not limited to, specific antibodies by selection from phage libraries, cloning of antibody cDNAs, and cloning of variable regions using adjacent constant regions as primers. Cloning of the variable regions of or the synthesis of oligonucleotides corresponding to the variable regions of any specific antibody. The cDNA can be modified at the 5 ′ and 3 ′ ends to generate restriction sites. As a result, the oligonucleotide linker can be used to clone the cDNA into a vector for cloning the HA cDNA. This can be done with or without a spacer sequence at the N-terminus or C-terminus. The cellular cDNA is cloned into a vector such as pPPC0005 (FIG. 2), pScCHSA, pScNHSA, or pC4: HSA. The complete expression cassette is then excised from such a vector and inserted into the plasmid pSAC35 to allow expression of the albumin fusion protein in yeast.

In the fusion proteins of the present invention, V _H and V _L, one of the following means or a combination thereof, can be linked: peptide linker between the C-terminus and N-terminus of the V _L of V _H; V _H and _{V L} is cleaved upon secretion, then self-associate, Kex2p protease cleavage site between the _{V H} and _{V L;} and, forming a disulfide bond between the _{V H} and _{V L,} with each other A cysteine residue at a position where it can be linked. An alternative option is to place _VH at the N-terminus of the HA or HA domain fragment and _VL at the C-terminus of the HA or HA domain fragment.

The albumin fusion protein secreted from the yeast can then be collected from the medium, purified and tested for its activity. A similar procedure applies for expression in mammalian cell lines, except that the expression cassette used uses a mammalian promoter, leader sequence and terminator (see Example 1). The expression cassette is then excised and inserted into a plasmid suitable for mammalian cell line transfection. Antibodies produced in this manner can be purified from the medium and tested for binding to the antigen using standard immunochemical methods.
(Example 55: Preparation of HA-cell adhesion molecule fusion protein)
The cDNA of the cell adhesion molecule of interest can be isolated by various means. Various means include, but are not limited to: RT-PCR or PCR using a series of overlapping synthetic oligonucleotide primers from a cDNA library, all using standard methods. The nucleotide sequences of known cell adhesion molecules are known and available in public databases such as GenBank. The cDNA can be modified at the 5 ′ and 3 ′ ends to generate restriction sites. As a result, the oligonucleotide linker can be used to clone the cDNA into a vector for cloning the HA cDNA. This can be done with or without a spacer sequence at the N-terminus or C-terminus. The cell adhesion molecule cDNA is cloned into a vector such as pPPC0005 (FIG. 2), pScCHSA, pScNHSA, or pC4: HSA. The complete expression cassette is then excised from such a vector and inserted into the plasmid pSAC35 to allow expression of the albumin fusion protein in yeast. The albumin fusion protein secreted from the yeast can then be collected from the medium, purified and tested for its biological activity. A similar procedure applies for expression in mammalian cell lines, except that the expression cassette used uses a mammalian promoter, leader sequence and terminator (see Example 1). The expression cassette is then excised and inserted into a plasmid suitable for mammalian cell line transfection.

Example 56: Preparation of inhibitors and peptides as HA fusion proteins (eg HA antiviral inhibitors, HA antibacterial inhibitors, HA enzyme inhibitors and HA antiallergic proteins)
The cDNA of the peptide of interest (eg, an antimicrobial peptide) can be obtained from a cDNA library by various means (RT-PCR and PCR using a series of overlapping synthetic oligonucleotide primers, all of which use standard methods). Including, but not exclusively). The cDNA can be altered at the 5 ′ and 3 ′ ends to create restriction sites so that the oligonucleotide linker can be used for cloning of the cDNA into a vector containing the HA cDNA. This can be at the N-terminus or C-terminus with or without a spacer sequence. The peptide cDNA is cloned into a vector such as pPPC0005 (FIG. 2), pScCHSA, pScNHSA or pC4: HSA, from which the complete expression cassette is then excised and inserted into plasmid pSAC35 for albumin fusion in yeast Allows protein expression. The albumin fusion protein secreted from yeast can then be collected and purified from the media and tested for its biological activity. For expression in mammalian cell lines, a similar procedure is employed except that the expression cassette used uses a mammalian promoter, leader sequence and terminator (see Example 1). This expression cassette is then excised and inserted into a plasmid suitable for mammalian cell line transfection.

Example 57: Preparation of targeted HA fusion protein
The cDNA of the protein of interest can be isolated from a cDNA library using standard molecular biology methods, or can be synthesized using several overlapping oligonucleotides. Appropriate nucleotides can be engineered in the cDNA to form convenient restriction sites and allow attachment of protein cDNA to albumin cDNA in a manner similar to that described for hGH. Targeted protein or peptide cDNA (eg, a peptide such as a single chain antibody or a nuclear localization signal that can direct the protein into the cell) can be fused to the other end of albumin. The protein of interest and targeting peptide are cloned into a vector that allows fusion with albumin cDNA (eg, pPPC0005 (FIG. 2), pScCHSA, pScNHSA, or pC4: HSA). In this manner, both the N-terminus and C-terminus of albumin are fused with other proteins. The fused cDNA is then excised from pPPC0005 and inserted into a plasmid such as pSAC35 to allow expression of the albumin fusion protein in yeast. All of the above procedures can be performed using standard methods of molecular biology. Albumin fusion protein secreted from yeast is recovered and purified from the media and tested for its biological and targeting activity using appropriate biochemical and biological tests.

Example 58: Preparation of HA-enzyme fusion
The cDNA of the enzyme of interest is obtained from a cDNA library, by RT-PCR, and by a series of overlapping synthetic oligonucleotides, including the following procedures, all using standard methods, but not exclusively: By PCR using primers). The cDNA can be modified at the 5 'and 3' ends to create restriction sites so that an oligonucleotide linker can be used to clone the cDNA into a vector containing the HA cDNA. This procedure can be done at the N-terminus or C-terminus with or without a spacer sequence. The enzyme cDNA is cloned into a vector such as pPPC0005 (FIG. 2), pScCHSA, pScNHSA, or pC4: HSA, from which the complete expression cassette is then excised and inserted into the plasmid pSAC35. Allows expression of albumin fusion proteins in The albumin fusion protein secreted from the yeast is then recovered from the medium and purified and tested for its biological activity. For expression in mammalian cell lines, a similar procedure applies (see Example 1), except that the expression cassette used uses a mammalian promoter, leader sequence, and terminator. This expression cassette is then excised and inserted into a plasmid suitable for transfection into a mammalian cell line.

(Example 59: Bacterial expression of albumin fusion protein)
A polynucleotide encoding an albumin fusion protein of the present invention containing a bacterial signal sequence is amplified using PCR oligonucleotide primers corresponding to the 5 'and 3' ends of the DNA sequence to synthesize an insert fragment. The primer used to amplify the polynucleotide encoding the insert preferably contains a restriction site (eg, BamHI and XbaI) at the 5 'end of the primer to clone the amplification product into an expression vector. . For example, BamHI and XbaI correspond to restriction enzyme sites in the bacterial expression vector pQE-9 (Qiagen, Inc., Chatsworth, CA). This plasmid vector consists of antibiotic resistance (Amp ^r ), bacterial origin of replication (ori), IPTG-regulatable promoter / operator (P / O), ribosome binding site (RBS), 6-histidine tag (6-His tag) ), And a restriction enzyme cloning site.

The pQE-9 vector is digested with BamHI and XbaI and the amplified fragment is ligated into the pQE-9 vector while maintaining the reading frame starting with bacterial RBS. The ligation mixture is then used to E. coli strain M15 / rep4, which expresses the lacI repressor and also confers kanamycin resistance (Kan ^r ) contains plasmid pREP4 in multiple copies (Quiagen, Inc.). Transformants are identified by their ability to grow on LB plates and ampicillin / kanamycin resistant colonies are selected. Plasmid DNA is isolated and confirmed by restriction analysis.

Clones containing the desired construct are grown overnight (O / N) in liquid culture in LB medium supplemented with both ampicillin (100 μg / ml) and kanamycin (25 μg / ml). A large volume of medium is inoculated using an O / N culture at a ratio of 1: 100 to 1: 250. Cells are grown until the optical density 600 (OD ⁶⁰⁰ ) is between 0.4 and 0.6. Next, IPTG (isopropyl-β-D-thiogalactopyranoside) is added at a final concentration of 1 mM. IPTG induces by inactivating the lacI repressor, leaving P / O empty, resulting in increased gene expression.

  Cells are grown for an additional 3-4 hours. Cells are then collected by centrifugation (6000 × g, 20 minutes). By agitation of the cell pellet in the chaotropic agent 6M guanidine HCl, or preferably 8M urea, and 2-mercaptoethanol at a concentration higher than 0.14M for 3-4 hours at 4 ° C. Solubilize (see, eg, Burton et al., Eur. J. Biochem. 179: 379-387 (1989)). Cell debris is removed by centrifugation and the supernatant containing the polypeptide is loaded onto a nickel-nitrilotriacetic acid (“Ni-NTA”) affinity resin column (available from QIAGEN, Inc. supra). Proteins with a 6 × His tag bind to Ni-NTA resin with high affinity and can be purified in a simple one-step procedure (for details, see The QIA expressionist (1995), QIAGEN, Inc., supra, checking).

  Briefly, the supernatant is loaded onto the column in 6M guanidine-HCl (pH 8). The column is first washed with 10 volumes of 6M guanidine-HCl, pH 8, then washed with 10 volumes of 6M guanidine-HCl, pH 6, and finally the polypeptide is eluted with 6M guanidine-HCl, pH 5.

  The purified protein is then regenerated by dialysis against phosphate buffered saline (PBS) or 50 mM sodium acetate, pH 6 buffer with 200 mM NaCl. Alternatively, the protein can be successfully refolded while immobilized on a Ni-NTA column. Exemplary conditions are as follows: regeneration using a linear 6M-1M urea gradient in 500 mM NaCl, 20% glycerol, 20 mM Tris / HCl pH 7.4 with protease inhibitors. Regeneration should be done for at least 1.5 hours. After regeneration, the protein is eluted by adding 250 mM imidazole. Imidazole is removed by a final dialysis step against 50 mM sodium acetate, pH 6 buffer with PBS or 200 mM NaCl. Purified protein is stored at 4 ° C. or frozen at −80 ° C.

  In addition to the above expression vectors, the present invention further includes an expression vector designated pHE4a (ATCC Accession No. 209645, deposited on February 25, 1998). This vector contains the phage operator and promoter elements operably linked to a polynucleotide encoding the albumin fusion protein of the invention (designated as pHE4a (ATCC Accession No. 209645, deposited 25 February 1998)). . This vector includes: 1) the neomycin phosphotransferase gene as a selectable marker, 2) E. coli. E. coli origin of replication, 3) T5 phage promoter sequence, 4) two lac operator sequences, 5) Shine-Dalgarno sequence, and 6) lactose operon repressor gene (lacIq). The origin of replication (oriC) is derived from pUC19 (LTI, Gaithersburg, MD). Promoter and operator sequences are synthesized.

  DNA can be inserted into pHE4a by restricting the vector with NdeI and XbaI, BamHI, XhoI or Asp718, running the restricted product on a gel, and isolating the larger fragment (the stuffer fragment is About 310 base pairs). DNA inserts are described for NdeI (5 ′ primer) and XbaI, BamHI, XhoI or Asp718 (3 ′ primer) according to PCR protocols described herein or otherwise known in the art. It is generated using PCR primers with the following restriction sites. The PCR insert is purified on a gel and restricted with a compatible enzyme. The insert and vector are ligated according to standard protocols.

  The engineered vector can be replaced in the protocol described above to express the protein in a bacterial system.

(Example 60: Expression of albumin fusion protein in mammalian cells)
The albumin fusion protein of the present invention can be expressed in mammalian cells. A typical mammalian expression vector contains a promoter element that mediates transcription initiation of mRNA, a protein coding sequence, and signals necessary for transcription termination and transcription polyadenylation. Additional elements include enhancers, Kozak sequences, and intervening sequences adjacent to donor and acceptor sites for RNA splicing. Highly efficient transcription is achieved by the early and late promoters of SV40, the long terminal repeats (LTR) of retroviruses (eg, RSV, HTLVI, HIVI), and the early promoter of cytomegalovirus (CMV). However, cellular elements can also be used (eg, the human actin promoter).

  Suitable expression vectors for use in the practice of the present invention include, for example, (pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), pBC12MI (ATCC 67109), pCMVSport. Vectors such as 0, and pCMVSPort 3.0 Mammalian host cells that can be used include, but are not limited to, human Hela cells, 293 cells, H9 cells, and Jurkat cells, mouse NIH3T3 and C127 cells, Cos1 cells, Cos7 cells and CV1 cells, quail QC1-3 cells, mouse L cells, and Chinese hamster ovary (CHO) cells. .

  Alternatively, the albumin fusion protein can be expressed in a stable cell line containing a polynucleotide encoding an albumin fusion protein integrated into a chromosome. Co-transfection with a selectable marker such as DHFR, gpt, neomycin, or hygromycin allows the identification and isolation of the transfected cells.

  The transfected polynucleotide encoding the fusion protein can be amplified to express large amounts of the encoded fusion protein. DHFR (dihydrofolate reductase) markers are useful for the generation of cell lines carrying the gene of interest in hundreds or thousands of copies. (See, eg, Alt et al., J. Biol. Chem. 253: 1357-1370 (1978); Hamlin et al., Biochem. Et Biophys. Acta, 1097: 107-143 (1990); Page et al., Biotechnology 9: 64-68 ( 1991)) Another useful selectable marker is the enzyme glutamine synthase (GS) (Murphy et al., Biochem J. 227: 277-279 (1991); Bebbington et al., Bio / Technology 10: 169-). 175 (1992)). Using these markers, mammalian cells are grown in selective media to select highly resistant cells. These cell lines contain the amplified gene integrated into the chromosome. Chinese hamster ovary (CHO) cells and NSO cells are often used for protein production.

  Plasmids pSV2-dhfr (ATCC Accession No. 37146), expression vector pC4 (ATCC Accession No. 209646), and pC6 (ATCC Accession No. 209647) are the rous sarcoma virus strong promoter (LTR) (Cullen et al., Molecular and Cellular Biology, 438-447 (March, 1985)), and CMV-enhancer (Boshart et al., Cell 41: 521-530 (1985)). Multiple cloning sites (eg, with restriction enzyme cleavage sites, BamHI, XhoI, and Asp718) facilitate cloning of the gene of interest. The vector also contains the 3 'intron of the rat preproinsulin gene, polyadenylation and termination signals, and the mouse DHFR gene under the control of the SV40 early promoter.

  Specifically, for example, plasmid pC6 is digested with appropriate restriction enzymes by procedures known in the art, and then dephosphorylated using bovine intestinal phosphatase. The vector is then isolated from a 1% agarose gel.

  A polynucleotide encoding an albumin fusion protein of the present invention is generated using techniques known in the art, and the polynucleotide is amplified using PCR techniques known in the art. If the naturally occurring signal sequence is used to produce the fusion protein of the invention, the vector does not require a second signal peptide. Alternatively, if a naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence (see, eg, International Publication No. WO 96/34891).

  The amplified fragment encoding the fusion protein of the invention is isolated from 1% agarose using a commercially available kit (“Geneclean”, BIO 101 Inc., La Jolla, Ca.). The fragment is then digested with the appropriate restriction enzymes and purified again in 1% agarose.

  The amplified fragment encoding the albumin fusion protein of the invention is then digested with the same restriction enzymes and purified on a 1% agarose gel. The isolated fragment and dephosphorylated vector are then ligated using T4 DNA ligase. Next, E.E. E. coli HB101 or XL-1 Blue cells are transformed and the bacteria containing the fragment inserted into plasmid pC6 are identified using, for example, restriction enzyme analysis.

  Chinese hamster ovary cells lacking an active DHFR gene are used for transfection. 5 μg of expression plasmid pC6 or pC4 is cotransfected with 0.5 μg of plasmid pSVneo using lipofection (Felgner et al., Supra). Plasmid pSV2-neo contains the Tn5-derived neo gene which encodes an enzyme conferring resistance to a group of antibiotics including G418, which is a dominant selectable marker. Cells are seeded in alpha minus MEM supplemented with 1 mg / ml G418. Two days later, cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng / ml methotrexate supplemented with 1 mg / ml G418. . After about 10-14 days, single clones are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks with different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). To do. Next, clones growing at the highest concentration of methotrexate are transferred to new 6-well plates containing still higher concentrations of methotrexate (1 μM, 2 μM, 5 μM, 10 mM, 20 mM). The same procedure is repeated until clones are obtained that grow at a concentration of 100-200 μM. Expression of the desired fusion protein is analyzed, for example, by SDS-PAGE and Western blot, or by reverse phase HPLC analysis.

(Example 61: Fusion of multiple fusion)
An albumin fusion protein (eg, a therapeutic protein (or fragment or variant thereof) fused to albumin (or a fragment or variant thereof) is further fused with another protein, resulting in a “multiple fusion protein”. obtain. These multiple fusion proteins can be used for various applications. For example, fusing the albumin fusion protein of the present invention with a His tag, HA tag, protein A, IgG domain, and maltose binding protein facilitates purification (eg, EP A 394, 827; Traunecker et al., Nature 331). : 84-86 (1988)). Nuclear localization signals fused to the polypeptides of the invention can target proteins to specific subcellular regions, while covalent heterodimers or homodimers are active in albumin fusion proteins. Can be increased or decreased. Furthermore, the fusion of an albumin fusion protein of the present invention with additional protein sequences can further improve the solubility and / or stability of the fusion protein. Said fusion protein may be obtained by using techniques known in the art or by routine modification and / or the following protocol (this outlines the fusion of a polypeptide with an IgG molecule). ) Can be modified.

  Briefly, the human Fc portion of an IgG molecule can be amplified by PCR using primers that span the 5 'and 3' ends of the sequence shown below. These primers should also have convenient restriction enzyme sites that facilitate cloning into an expression vector, preferably a mammalian or yeast expression vector.

  For example, when pC4 (ATCC accession number 209646) is used, the human Fc portion can be linked to the BamH 'cloning site. Note that the 3'BamH 'site should be destroyed. The vector containing the human Fc portion is then recut into BamH ′, linearized into the vector, and a polynucleotide encoding the albumin fusion protein of the invention (generated and isolated using techniques known in the art) ) Is linked to this BamH ′ site. It should be noted that the polynucleotide encoding the fusion protein of the invention is cloned without a stop codon, otherwise an Fc-containing fusion protein is not produced.

If the natural signal sequence is used to produce the albumin fusion protein of the invention, pC4 does not require a second signal peptide. Alternatively, if the native signal sequence is not used, the vector can be modified to include a heterologous signal sequence (see, eg, International Publication No. WO 96/34891).
Human IgG Fc part:

（実施例６２：アルブミン融合タンパク質由来の抗体の産生）
（ハイブリドーマ技術）
本発明のアルブミン融合タンパク質および本発明のアルブミン融合タンパク質の１部分（例えば、融合タンパク質の治療的タンパク質部分またはアルブミン部分）に結合する抗体は、様々な方法（Ｃｕｒｒｅｎｔ Ｐｒｏｔｏｃｏｌｓ、第２章を参照のこと）によって調製され得る。そのような方法の１例として、本発明のアルブミン融合タンパク質または本発明のアルブミン融合タンパク質の１部分の調製物が、調製され、実質的に天然の汚染物を含まないように精製される。その後、そのような調製物はより大きな比活性を有するポリクローナル血清を生産するために、動物に導入される。

(Example 62: Production of antibody derived from albumin fusion protein)
(Hybridoma technology)
Antibodies that bind to an albumin fusion protein of the invention and a portion of an albumin fusion protein of the invention (eg, a therapeutic protein portion or an albumin portion of a fusion protein) can be found in various ways (Current Protocols, Chapter 2). ). As an example of such a method, an albumin fusion protein of the invention or a preparation of a portion of an albumin fusion protein of the invention is prepared and purified to be substantially free of natural contaminants. Such preparations are then introduced into animals to produce polyclonal sera with greater specific activity.

  Monoclonal antibodies specific for the albumin fusion protein of the present invention or a portion of the albumin fusion protein of the present invention are prepared by hybridoma technology (Kohler et al., Nature 256: 495 (1975); Kohler et al., Eur. J. Immunol. 6: 511). Kohler et al., Eur. J. Immunol. 6: 292 (1976); Hammerling et al., Monoclonal Antibodies and T-cell Hybridomas, Elsevier, NY, pp. 563-681 (1981)). Prepared. In general, an animal (preferably a mouse) is immunized with an albumin fusion protein of the invention or a portion of an albumin fusion protein of the invention. Spleen cells from such mice are extracted and fused with an appropriate myeloma cell line. Any suitable myeloma cell line can be used in accordance with the present invention; however, the use of the parent myeloma cell line (SP2O) (available from ATCC) is preferred. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium and then cloned by limiting dilution as described by Wands et al. (Gastroenterology 80: 225-232 (1981)). . Hybridoma cells obtained through such selection are then assayed to identify clones that secrete antibodies that can bind to an albumin fusion protein of the invention or a portion of an albumin fusion protein of the invention.

  Alternatively, additional antibodies that can bind to an albumin fusion protein of the invention or a portion of an albumin fusion protein of the invention can be produced by a two-step procedure using an anti-idiotype antibody. Such methods take advantage of the fact that antibodies are themselves antigens, so it is possible to obtain antibodies that bind to secondary antibodies. According to this method, a protein specific for an antibody is used to immunize an animal, preferably a mouse. The spleen cells of such animals are then used to produce hybridoma cells that bind to an antibody specific for the albumin fusion protein of the invention (or a portion of the albumin fusion protein of the invention). Screened to identify clones that produce antibodies whose ability can be blocked by a fusion protein of the invention or a portion of an albumin fusion protein of the invention. Such antibodies include anti-idiotypic antibodies against antibodies specific for the fusion protein of the invention (or a portion of the albumin fusion protein of the invention) and the fusion protein of the invention (or the albumin fusion protein of the invention). Used to immunize animals for the purpose of inducing further formation of antibodies specific for one part).

  For in vivo use of antibodies in humans, antibodies are “humanized”. Such antibodies can be produced using a genetic construct derived from a hybridoma cell that produces the monoclonal antibody described above. Methods for producing chimeric and humanized antibodies are known in the art and discussed herein (reviewed in Morrison, Science 229: 1202 (1985); Oi et al., BioTechniques 4: 214 (1986). Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO8601533; Robinson et al., International Publication No. 8702671; Boulianne et al. : 268 (1985)).

(Isolation of an albumin fusion protein of the present invention or an antibody fragment derived from a library of scFvs or a portion of the albumin fusion protein of the present invention)
The natural V gene isolated from human PBL has been constructed into a library of antibody fragments containing reactivity to an albumin fusion protein of the invention or a portion of an albumin fusion protein of the invention. There, the donor may or may not be exposed. (See, eg, US Pat. No. 5,885,793, incorporated herein by reference in its entirety)
(Rescue library)
The scFvs library is constructed from human PBL RNA as described in International Publication No. WO92 / 01047. Approximately 10 ⁹ E. coli harboring phagemids are used to rescue phage displaying antibody fragments. E. coli was used and seeded in 2 × TY (2 × TY-AMP-GLU) (50 ml) containing 1% glucose and 100 μg / ml ampicillin. D. Grow with shaking until is 0.8. Of this culture, 5 ml is used to inoculate 2 × TY-AMP-GLU (50 ml), 2 × 10 8 TU delta gene 3 helper (M13 delta gene ′ ″, see International Publication No. WO 92/01047) And the medium is incubated for 45 minutes at 37 ° C. without shaking, and then for 45 minutes at 37 ° C. with shaking. The culture solution is 4000 r. p. m. For 10 minutes and the pellet is resuspended in 2 × TY (2 l) containing 100 μg / ml ampicillin and 50 μg / ml kanamycin and grown overnight. The phage is prepared as described in International Publication No. WO 92/01047.

The M13 delta gene III is prepared as follows: The M13 delta gene III helper phage does not encode the gene III protein, so the phage displaying the antibody fragment (mid) has large binding to the antigen. Have the ability. Infectious M13 delta gene III particles are made by the expansion of helper phage in cells carrying pUC19 derivatives that provide wild type gene III protein during phage morphogenesis. The culture is incubated for 1 hour at 37 ° C. without shaking and then for another hour at 37 ° C. with shaking. Cells were spun down (IEC-Centra 8,400 rpm) for 10 minutes and 2 × TY broth (2 × TY-AMP-KAN) (300 ml) containing 100 μg / ml ampicillin and 25 μg / ml kanamycin. And grown overnight at 37 ° C. with shaking. Phage particles were generated from the culture medium by two rounds of PEG precipitation (Sambrook et al., 1990), collected, resuspended in 2 ml PBS, passed through a 0.45 μm filter to a final concentration of about 10 ^13. Unit / ml (ampicillin resistant clone).

(Library panning)
Immunotubes (Nunc) were plated overnight in PBS with 100 μg / ml or 10 μg / ml of the albumin fusion protein of the invention or a portion of the albumin fusion protein of the invention (4 ml). Tubes are blocked with 2% Marvel-PBS for 2 hours at 37 ° C. and then washed 3 times with PBS. Approximately 10 ¹³ TU of phage is applied to the tube and incubated for 30 minutes at room temperature while lying on the top and bottom of the turntable. It is then left to stand for an additional 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and 10 times with PBS. The phage should be added with 1 ml of 100 mM triethylamine and rotated on and under the turntable for 15 minutes (after which the solution is immediately neutralized with 0.5 ml of 1.0 M Tris-HCl, pH 7.4). It is eluted with. Subsequently, the phage was incubated with bacteria at 37 ° C. for 30 minutes with 10 ml of medium-logging E. coli. used to infect E. coli TG1. Thereafter, this E.E. E. coli is plated on TYE plates containing 1% glucose and 100 μg / ml ampicillin. The resulting bacterial library is then rescued by delta gene 3 helper phage at a later selection stage, as described above for preparing the phage. Thereafter, in this process, affinity purification is repeated a total of 4 times, where the tube is washed 20 times with PBS, 0.1% Tween-20, 20 times with PBS in the third and fourth time.

(Characteristics of the conjugate)
The phage eluted from the third and fourth rounds of selection A soluble scFv is produced from a single colony by the assay used to infect E. coli HB2151 (Marks et al., 1991). The ELISA method was performed by microtiter replates coated with either 10 pg / ml of the albumin fusion protein of the present invention or a portion of the albumin fusion protein of the present invention in 50 mM bicarbonate buffer (pH 9.6). Implemented. ELISA positive clones are further characterized by PCR fingerprinting (see, eg, International Publication No. WO 92/01047) and subsequent sequencing. These ELISA positive clones are also known in the art (eg, epitope mapping, binding affinity, receptor signaling, ability to block or competitively inhibit antibody / antigen binding, and competitive agonists). Activity or antagonist activity).

Example 63: Method of treatment using ex vivo gene therapy
One method of gene therapy transplants fibroblasts, which can express the albumin fusion protein of the invention into a patient. Generally, fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in tissue culture medium and separated into small pieces. This tissue blob is placed on the wet surface of the tissue culture flask and about 10 pieces are placed in each flask. Shake the flask up and down, close tightly and leave at room temperature overnight. After 24 hours at room temperature, the flask is inverted and the tissue mass remains fixed at the bottom of the flask and fresh medium (eg, Ham's F12 medium (containing 10% FRS, penicillin and streptomycin)) is added. . The flask is then incubated at 37 ° C. for about 1 week.

  At this time, fresh medium is added and then replaced every few days. After an additional 2 weeks of culture, a fibroblast monolayer appears. This monolayer is trypsinized and scaled to a larger flask.

  pMV-7 (Lirschmeier, PT et al., DNA, 7: 219-25 (1988)) (adjacent the Moloney murine sarcoma virus n-terminal repeat) was digested with EcoRI and HindIII, followed by Treat with bovine intestinal phosphatase. The linear vector is fractionated on an agarose gel and purified using glass beads.

  A polynucleotide encoding an albumin fusion protein of the invention can be generated using techniques known in the art and can be amplified using PCR primers, which correspond to 5 'and 3' end sequences. If necessary, it has appropriate restriction sites and, if necessary, start / stop codons. Preferably, the 5 'primer comprises an EcoRI site and the 3' primer comprises a HindIII site. In the presence of T4 DNA ligase, an equivalent amount of Moloney murine sarcoma virus linear spine and amplified EcoRI and HindIII fragments are added together. The resulting mixture is maintained under conditions suitable for ligation of the two fragments. The ligation mixture is then used to transform bacterial HB101, which is then plated on agar (including kanamycin) in order to confirm that the vector has the gene of interest properly inserted. .

  Amphoteric pA317 or GP + am12 packaged cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) (containing 10% calf serum (CS), penicillin and streptomycin). The MSV vector containing this gene is then added to the medium and the packaged cells are transformed with the vector. Here, the package cell generates a ring teacher virion (including a gene, where the package cell is referred to as a producer cell).

  Fresh medium is added to the transformed producer cells, which is subsequently recovered from a 10 cm plate of confluent producer cells. Consumed medium (containing infectious virus particles) is filtered through a Millpore filter to remove dissociated producer cells, and this medium is then used to allow the fibers to infect the cells. The media is removed from the fibroblast semi-confluent plate and rapidly replaced with media from producer cells. This medium is removed and replaced with fresh medium. If the virus titer is high, virtually all fibroblasts are infected and no selection is required. If the titer is very low, it is necessary to use a retroviral vector with a selectable marker (eg, neo or his). Once the fibroblasts are efficiently infected, the fibroblasts are analyzed to determine if albumin fusion protein has been produced.

  The engineered fibroblasts are then transplanted into the host after growing to confluence alone or on cytodex 3 microcarrier beads.

Example 64: Method of treatment using in vivo gene therapy
Another aspect of the invention is to treat disorders, diseases and conditions using in vivo gene therapy methods. Gene therapy methods relate to the introduction of naked nucleic acids (DNA, RNA, and antisense DNA or RNA) encoding the albumin fusion protein of the invention into animals. A polynucleotide encoding an albumin fusion protein of the invention can be operably linked (ie, associated) with a promoter or any other genetic element required for expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques and methods are known in the art. For example, W090 / 11092, W098 / 11779; U.S. Patent Nos. 5,693,622, 5,705,151, and 5,580,859; Tabata et al. Res. 35 (3): 470-479 (1997); Chao et al., Pharmacol. Res. 35 (6): 517-522 (1997); Wolff, Neuromuscul. Disorder. 7 (5): 314-318 (1997); Schwartz et al., Gene Ther. 3 (5): 405-411 (1996); Tsurumi et al., Circulation 94 (12): 3281-3290 (1996) (incorporated herein by reference).

  The polynucleotide construct can be delivered by any method that delivers injectable material to the cells of the animal (eg, injection into the interstitial space of the tissue (heart, muscle, skin, lung, liver, intestine, etc.)). The polynucleotide construct can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.

  The term “naked” polynucleotide, DNA or RNA refers to any delivery vehicle that acts to assist, enhance or promote cell entry (such as viral sequences, viral particles, liposomal formulations, lipofectin or precipitants). An array that does not contain However, a polynucleotide encoding an albumin fusion protein of the invention can also be delivered in a liposomal formulation (eg, Felgner PL et al. (1995) Ann. NY Acad. Sci. 772: 126-139 and Abdallah B (1995) Biol. Cell 85 (1): as taught in 1-7), these can be prepared by methods well known to those skilled in the art.

  Polynucleotide vector constructs used in gene therapy methods are preferably constructs that do not integrate into the host genome or contain sequences that allow replication. Any strong promoter known to those skilled in the art can be used to drive the expression of DNA. Unlike other gene therapy techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the temporary nature of polynucleotide synthesis in the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells and provide production of the desired polypeptide in a period of up to 6 months.

  The polynucleotide construct can be delivered to the interstitial space of the following tissues in the animal: muscle, skin, brain, liver, treasury, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, Gallbladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue. The interstitial space of these tissues is intercellular fluid, mucopolysaccharide matrix between reticulofibers of organ tissue, elastic fibers of vascular or chamber walls, collagen fibers of fibrous tissue, or connective tissue covering muscle cells Including the same matrix within or within the bone void. It is likewise the space occupied by circulating fluid in the circulating plasma and lymph channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They can be conveniently delivered by injection into tissues containing these cells. They are preferably delivered and expressed continuously into differentiated, non-dividing cells, but delivery and expression is non-differentiated cells or cells that are not fully differentiated (eg, blood stem cells Or skin fibroblasts). In vivo, myocytes are particularly competent in the capacity for polynucleotide uptake and expression.

  For naked polynucleotide injections, effective dosages of DNA or RNA range from about 0.05 g / kg body weight to about 50 mg / kg body weight. Preferably, the dosage is from about 0.005 mg / kg to about 20 mg / kg, and more preferably from about 0.05 mg / kg to about 5 mg / kg. Of course, as those skilled in the art will appreciate, this dosage will vary depending on the tissue to be injected. Appropriate and effective dosages of the nucleic acid sequences can be readily determined by one skilled in the art and can depend on the condition being treated and the route of administration. The preferred route of administration is by the parenteral route of injection into the interstitial space of the tissue. However, other parenteral routes (eg, inhalation of aerosol formulations, particularly for delivery to lung or bronchial tissue, throat or nasal mucosa) may also be used. In addition, naked polynucleotide constructs can be delivered to the artery during angioplasty with a catheter used in the procedure.

  The in vivo dose response effect of polynucleotide injected intramuscularly is determined as follows. A template DNA suitable for the production of mRNA encoding a polypeptide of the invention is prepared according to standard recombinant DNA methodology. Template DNA (which can be either circular or linear) is used as naked DNA or complexed with liposomes. The mouse quadriceps are then injected with various amounts of template DNA.

  Five to six week old female and male Balb / C mice are anesthetized by intraperitoneal injection with 0.3 ml of 2.5% Abertin. A 1.5 cm incision is made in the anterior thigh and the quadriceps is directly visualized. Template DNA was injected through a 27 cc needle of a 1 cc syringe for 1 minute with 0.1 ml carrier at about 0.5 cm and a depth of about 0.2 cm from the distal injection site of the muscle into the knee. For future localization, a suture is placed at the injection site and the skin is closed with a stainless steel clip.

  After an appropriate incubation time (eg 7 days), a muscle extract is prepared by incising the entire quadriceps. Each fifth section of each quadriceps is histochemically stained for protein expression. The time course for the fusion protein can be performed in a similar manner, except that the quadriceps from different mice are collected at different times. The persistence of intramuscular DNA after injection can be determined by Southern blot analysis after preparation of total cellular DNA and HIRT supernatants from injected and control mice. The results of the above experiments in mice can be used to interpolate appropriate parameters and other treatment parameters using naked DNA in humans and other animals.

(Example 65: Transgenic animal)
The albumin fusion proteins of the present invention can also be expressed in transgenic animals. Animals of any species include, but are not limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs, small pigs, goats, sheep, cows and non-human primates (eg baboons, monkeys and chimpanzees) ) Can be used to generate transgenic animals. In certain embodiments, the fusion proteins of the invention are expressed in humans as part of a gene therapy protocol using techniques described herein or otherwise known in the art.

Any technique known in the art can be used to introduce a polynucleotide encoding an albumin fusion protein of the invention into an animal to generate a primary line of transgenic animals. Such techniques include, but are not limited to, pronuclear microinjection (Patterson et al., Appl. Microbiol. Biotechnol. 40: 691-698 (1994); Carver et al. , Biotechnology (NY) 11: 1263-1270 (1993); Wright et al., Biotechnology (NY) 9: 830-834 (1991); and Hoppe et al., US Pat. No. 4,873,191 (1989)); germline (Van der Putten et al., Proc. Natl. Acad. Sci., USA 82: 6148-6152 (1985)), retrovirus-mediated gene transfer into blasts or germ cells; embryonic stem Gene targeting in cells (Thompson et al., Cell 56: 313-321 (1989)); electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol. 3: 1803-1814 (1983)); Introduction of a polynucleotide of the invention to be used (see, eg, Ulmer et al., Science 259: 1745 (1993)); introduction of nucleic acid constructs into embryonic pleural potential stem cells and transfer of stem cells into blasts; and semen Mediated gene transfer (Lavitrano et al., Cell 57: 717-723 (1989)); For a review of such techniques, see Gordon, “Transgenic Animals,” Intl. Rev. Cytol. 115: 171-229 (1989), which is hereby incorporated by reference in its entirety.

  Any technique known in the art (e.g., cultured embryonic embryos induced to dormancy, or nuclear transfer of nuclei from adult cells to enucleated oocytes (Campell et al., Nature 380: 64-66 (1996); Wilmut et al., Nature 385: 810-813 (1997))) can be used to generate transgenic clones comprising a polynucleotide encoding an albumin fusion protein of the invention.

  The present invention relates to transgenic animals that have polynucleotides encoding the albumin fusion protein of the invention throughout their cells, as well as animals that are not whole cells but possess some of these polynucleotides (ie, mosaic animals or chimeric animals). )I will provide a. A transgene can be integrated as a single transgene or as multiple copies (eg, head-to-head or head-to-tail connections) in a concatamer. This transgene can also be selectively introduced into specific cell types, for example by following the teachings of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA 89: 6232-6236 (1992)). And can be activated. The regulatory sequences required for cell type specific activation will depend on the particular cell type of interest and will be apparent to those skilled in the art. Gene targeting is preferred when it is desired that the polynucleotide encoding the fusion protein of the invention be integrated into the therapeutic protein portion or the chromosomal site of the endogenous gene corresponding to the albumin fusion protein of the invention. Briefly, when such techniques are utilized, a vector containing several nucleotide sequences homologous to an endogenous gene can be transferred to the nucleotide sequence of the endogenous gene via homologous recombination with a chromosomal sequence. Designed for the purpose of integration and destruction of its function. Transgenes can also be selectively introduced into a particular cell type, and thus are endogenous only in that cell type, for example by following the teachings of Gu et al. (Gu et al., Science 265: 103-106 (1994)). The gene can be inactivated. The regulatory sequences required for such cell-specific inactivation will depend on the particular cell type of interest and will be apparent to those skilled in the art.

  Once transgenic animals have been generated, recombinant gene expression can be assayed using standard techniques. Initial screening can be accomplished by Southern blot analysis or PCR techniques to analyze animal tissue and verify that integration of the polynucleotide encoding the fusion protein of the invention has occurred. The level of mRNA expression of a polynucleotide encoding the fusion protein of the invention in the tissue of a transgenic animal can also be assessed using techniques including but not limited to: tissue samples obtained from animals Northern blot analysis, in situ hybridization analysis, and reverse transcriptase PCR (rt-PCR). Samples of fusion protein expressing tissue can also be assessed immunocytochemically or immunohistochemically using antibodies specific for the fusion protein.

  Once founder animals are generated, they can be bred, inbred, crossbred, or crossbred to produce colonies of specific animals. Examples of such breeding strategies include, but are not limited to: cross-breeding founder animals with more than one integration site to establish separate lines; Mating to generate compound transgenic bodies that express the transgene at higher levels due to the effect of further expression of each transgene; so as to generate heterozygous animals at a given integration site Crossing heterozygous transgenic animals to reinforce expression and eliminate the need for screening animals by DNA analysis; crossing separate heterozygous lines to compound heterozygous lines or homozygotes As well as the transgene (ie, the present invention) against a separate background appropriate to the experimental model of interest. Breed to place polynucleotide) encoding albumin fusion proteins.

  The transgenic animals of the present invention have uses including, but not limited to, the following: animal model systems useful for estimating the biological function of the fusion proteins of the present invention as well as treatment of the fusion proteins of the present invention. Study of protein and / or albumin components, conditions and / or disorders associated with abnormal expression, and screening for compounds effective to ameliorate such conditions and / or disorders.

Example 66: Assay to detect stimulation or proliferation of B cells and differentiation
Generation of a functional humoral immune response requires soluble and cognate signaling between B-linked cells and their microenvironment. The signal can provide either a positive stimulus that allows the B-linked cell to sustain its programmed development or a negative stimulus that directs the cell to stop its current developmental pathway. To date, many stimulatory and inhibitory signals have been detected in B cell responses (IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-13, IL-14 and It has been found to affect IL-15). Interestingly, these signals are weak effectors by themselves, but when combined with various costimulatory proteins, activation, proliferation, differentiation, homing, tolerance, and tolerance among B cell populations. Can induce death.

  One of the best studied classes of B cell costimulatory proteins is the TNF superfamily. In this family, CD40, CD27, and CD30 with their respective ligands CD154, CD70, and CD153 have been found to modulate a variety of immune responses. Assays that allow detection and / or observation of the proliferation and differentiation of these B cell populations and their precursors are valuable tools for determining the effects of various proteins on these B cell populations in terms of proliferation and differentiation It is. The two assays listed below are designed to allow detection of differentiation, proliferation, or inhibition of B cell populations and their precursors.

  In Vitro Assay—Albumin fusion proteins of the invention (including therapeutic protein fragments or variants and / or albumin or various albumin fragments or variants) are activated, proliferated, differentiated in B cell populations and their precursors. Can be evaluated for their ability to induce inhibition and / or death. The activity of the albumin fusion protein of the present invention on purified human tonsil B cells (measured quantitatively for dosages in the range of 0.1-10,000 ng / mL) was measured simultaneously with standard B lymphocytes. Evaluated in a stimulation assay, where purified tonsillar B cells are cultured in the presence of either formalin-fixed Staphylococcus aureus Cowan I (SAC) or immobilized anti-human IgM antibody as the prime agent. The second signal (eg, IL-2 and IL-15) synergizes with the cross-linking of SAC and IgM to induce B cell proliferation as measured by tritiated thymidine incorporation. New synergists can be readily identified using this assay. This assay involves isolating human tonsil B cells by deficiency of magnetic beads (MACS) or CD3 positive cells. The resulting cell population is greater than 95% B cells as assessed by CD45R (B220) expression.

Various dilutions of each sample were plated into individual wells of a 96-well plate, which was added to 150 μL of total volume of culture medium (RPMI 1640, 10% FBS, 5 × 10 ⁻⁵ M 2ME, 100 U / Add 10 ⁵ B cells suspended in a dilution of ml penicillin, 10 ug / ml streptomycin, and 10 ⁻⁵ SAC). Growth or inhibition is quantified by initiating a 20 hour pulse (1 uCi / well) with 3H-thymidine (6.7 Ci / mM) 72 hours after element addition. Positive and negative controls are IL2 and medium, respectively.

  In vivo assay-buffer only twice daily or albumin fusion protein of the present invention (including therapeutic protein fragments or variants and / or albumin or various albumin fragments or variants) in BALB / c mice (I.p.). Mice receive this treatment for 4 consecutive days, at which time they are sacrificed and various tissues and sera are collected for analysis. Comparison of H & E sections from normal spleen and spleens treated with the albumin fusion protein of the invention results in the activity of the fusion protein on spleen cells (eg, diffusion of peripheral arterial lymph sheath and / or nucleation of red pulp) A significant increase in voice) was identified, which may indicate activation of differentiation and proliferation of the B cell population. Using immunohistochemical studies with the B cell marker anti-CD45R (B220), any physiological changes to spleen cells (eg, spleen segregation) are loosely defined that infiltrate established T cell regions. Determine whether it is due to increased B cell presentation within the B cell region.

  Using flow cytometric analysis of spleens from mice treated with albumin fusion protein, does albumin fusion protein increase the ratio of ThB +, CD45R (B220) dull B cells over that observed in control mice? Indicates whether or not.

  Similarly, the predicted result of increased mature B cell presentation in vivo is a relatively increased plasma Ig titer. Thus, plasma IgM and IgA levels are comparable between buffer and fusion protein treated mice.

Example 67: T cell proliferation assay
A CD3-induced proliferation assay is performed on PBMC and measured by ³ H-thymidine incorporation. This assay is performed as follows. 96 well plates were coated overnight at 4 ° C. with 100 μl / well mAb (HIT3a, Pharmingen) against CD3 or isotype matched control mAb (B33.1) (1 μg in 0.05 M carbonate buffer). / Ml, pH 9.5), then washed 3 times with PBS. PBMCs were isolated from human peripheral blood by F / H gradient centrifugation and various concentrations of the albumin fusion protein of the invention (fragments or variants of the therapeutic protein and in various concentrations in RPMI (including 10% FCS and P / S) and Add to quadruplicate wells (5 × 10 ⁴ wells) of mAb-coated plates in the presence of (or containing albumin or various albumin fragments or variants) (total volume 200 μl). The relevant protein buffer and medium alone are controls. After 48 hours of incubation at 37 ° C., rotate at 1000 rpm for 2 minutes and remove 100 μl of supernatant, if an effect on proliferation is observed—for measurement of IL-2 (or other cytokines) — Store at 20 ° C.
100 μl of medium (0.5 uCi ³ H-thymidine) is supplemented to the wells and incubated at 37 ° C. for 18-24 hours. Wells are collected and ³ H-thymidine incorporation is used as a measure of proliferation. Anti-CD3 alone is a positive control for proliferation. IL-2 (100 U / ml) is also used as a control, which enhances proliferation. A control antibody that does not induce T cell proliferation is used as a negative control for the effect of the fusion protein of the invention.

Example 68: MHC class II, expression of costimulatory and cell adhesion molecules, and the effect of the fusion protein of the invention on cell differentiation of monocytes and monocyte-derived human dendritic cells
Dendritic cells are generated by expansion of proliferative progenitors found in peripheral blood: adherent PBCM or elutriated monocyte fractions together with GM-CSF (50 ng / ml) and EL-4 (20 ng / ml) Incubate for 10 days. These dendritic cells have the characteristic phenotype of immature cells (expression of CD1, CD80, CD86, CD40 and MHC class II antigens). Treatment with an activator such as TNF-α allows for rapid surface phenotypes (NHC class I and class II, increased expression of costimulatory and cell adhesion molecules, FCγRII downregulation, CD83 upregulation). Make a change. These changes are associated with enhanced antigen presentation capacity and dendritic cell maturation.

  FACS analysis of surface antigen is performed as follows. Cells are treated with increasing concentrations of the albumin fusion protein of the invention or LPS (positive control) for 1-3 days, washed with PBS containing 1% BSA and 0.02 mM sodium azide, then appropriate FITC labeling Incubate with conjugated monoclonal antibody or PE labeled monoclonal antibody (1:20 dilution) at 4 ° C. for 30 minutes. After further washing, the labeled cells are analyzed by flow cytometry (FACScan (Becton Dickinson)).

(Influence of cytokine production)
Cytokines produced by dendritic cells (particularly IL-12) are important in inhibiting T cell-dependent immune responses. IL-2 strongly influences the development of Th1 helper T cell immune responses and induces cytotoxic T cell functions and NK cell functions. An ELISA is used to measure IL-12 release as follows. Dendritic cells (10 ⁶ / ml) are treated with increasing concentrations of the albumin fusion protein of the invention for 24 hours. LPS (100 ng / ml) is added to the cell culture as a positive control. The supernatants from these cell cultures are then collected and analyzed for IL-12 content using a commercially available ELISA kit (eg, R & D Systems (Minneapolis, Minn.)). Use the standard protocol attached to the kit.

(Influence of expression of MHC class II, costimulatory molecules and adhesion molecules)
Three major families of cell surface antigens can be observed in monocytes: adhesion molecules, molecules involved in antigen presentation, and Fc receptors. Regulation of expression of HMC class II antigens and other co-expressed molecules (eg, B7 and ICAM-1) can result in changes in monocyte antigen-presenting ability and ability to induce T cells. Increased Fc receptor expression may be associated with improved monocyte cytotoxic activity, cytokine release and phagocytosis.

  FACS analysis is used to test the expressed antigen as follows. Monocytes are treated with increasing concentrations of the albumin fusion protein of the invention or LPS (positive control) for 1-5 days, washed with PBS containing 1% BSA and 0.02 mM sodium azide, then appropriate FITC Incubate with labeled monoclonal antibody or PE labeled monoclonal antibody (1:20 dilution) at 4 ° C. for 30 minutes. After further washing, the labeled cells are analyzed by flow cytometry (FACScan (Becton Dickinson)).

(Monocyte activation and / or enhanced survival)
Assays for molecules that activate (or inactivate) monocytes and / or enhance monocyte survival (or decrease monocyte survival) are known in the art, and the molecules of the invention are monocytes. Can be routinely adapted to determine whether it functions as an inhibitor or activator. The albumin fusion proteins of the invention can be screened using the three assays described below. For each of these assays, peripheral blood mononuclear cells (PBMC) are purified from a single donor leukopack (American Red Cross, Baltimore, MD) by centrifugation through Histopaque gradient (Sigma). Monocytes are isolated from PBMC by countercurrent centrifugation elutriation.

(Monocyte survival assay)
Human peripheral blood monocytes gradually die when cultured in the absence of serum or other stimuli. Monocyte death is the result of an internal regulatory process (apoptosis). The addition of an activator such as TNF-α to the culture dramatically improves cell survival and protection from DNA fragmentation. Propidium iodide (PI) staining is used to measure apoptosis as follows. Monocytes are cultured in polypropylene tubes for 48 hours in serum-free medium (positive control), in the presence of 100 ng / ml TNF-α (negative control), and in the presence of various concentrations of the fusion protein to be tested. To do. Cells are suspended in PBS containing PI at a final concentration of 5 μg / ml at a concentration of 2 × 10 ⁶ cells / ml and then incubated at room temperature for 5 minutes prior to FACScan analysis. PI uptake has been demonstrated to correlate with DNA fragmentation in this experimental paradigm.

(Effect of cytokine release)
An important function of monocytes / macrophages is their regulatory activity on other cell populations of the immune system via cytokine release following stimulation. An ELISA to measure cytokine release is performed as follows. Human monocytes are cultured at a density of 5 × 10 ⁵ cells / ml and increasing concentrations of the albumin fusion protein of the invention, and under the same conditions but in the absence of this fusion protein. For IL-2 production, these cells are primed overnight with IFN (100 U / ml) in the presence of the fusion protein. LPS (10 ng / ml) is then added. Conditioned media is collected after 24 hours and stored frozen until use. The measurement of TNF-α, IL-10, MCP-1 and IL-8 was then performed using a commercially available ELISA kit (eg, R & D Systems (Minneapolis, Minn.)) To the standard protocol attached to the kit. Use to implement.

(Oxidative burst)
Purified monocytes are plated in 96 wells at 2-1 x 10 ⁵ cells / well. Increasing concentrations of the fusion protein of the invention are added to the wells in a total volume of 0.2 ml culture medium (RPMI 1640 + 10% FCS, glutamine and antibiotics). After 3 days incubation, the plates are centrifuged and the medium is removed from the wells. For macrophage monolayers, 0.2 ml of phenol red solution (140 mM NaCI, 10 mM potassium phosphate buffer (pH 7.0), 5.5 mM dextrose, 0.56 mM phenol red and 19 U / ml HRPO) per well. Add with stimulant (200 nM PMA). The plates are incubated for 2 hours at 37 ° C. and the reaction is stopped by adding 1N NaOH (20 μl) per well. Read absorbance at 610 nm. To measure the amount of H ₂ O ₂ produced by macrophages, a standard curve of H ₂ O ₂ solution with a known molar concentration is performed for each experiment.

(Example 69: Biological influence of the fusion protein of the present invention)
(Astrocyte and neural assay)
The albumin fusion proteins of the invention can be tested for activity in promoting cortical neuron survival, neurite outgrowth, or phenotypic differentiation, and for induction of proliferation of glial fibrillary acidic protein immunoprotective cells, astrocytes. Cortical cell selection for bioassays is based on the ubiquitous expression of FGF-1 and FGF-2 in the cortical structure and the promotion of cortical neuronal survival by previously reported FGF-2 treatment. For example, a thymidine incorporation assay can be used to elucidate the activity of an albumin fusion protein of the invention in these cells.

  Furthermore, previous reports describing the biological effects of FGF-2 (basic FGF) in cortical or hippocampal neurons in vitro have shown that both of these increase neuronal survival and neurite outgrowth. (Wilkeke et al., “Fibroblast growth factor promotes survival of dissociated hippocampal neurons and enhances neuroextension.”, Proc. Natl. 30. Proc. Natl. However, reports from experiments conducted on PC-12 cells show that these two reactions do not necessarily mean the same thing. Suggests that it may depend not only on the tested FGF but also on the receptors expressed on the target cells, using a primary cortical neuron culture paradigm to induce neurite outgrowth of the albumin fusion protein of the invention The ability can be compared to the response achieved with FGF-2 using, for example, a thymidine incorporation assay.

(Fibroblast assay and endothelial cell assay)
Human lung fibroblasts are obtained from Clonetics (San Diego, Calif.) And maintained in growth media from Clonetics. Dermal microvascular endothelial cells are obtained from Cell Applications (San Diego, Calif.). For proliferation assays, human lung fibroblasts and dermal microvascular endothelial cells can be cultured at 5,000 cells / well for 1 day in 96-well plate growth medium. The cells are then incubated for 1 day in 0.1% BSA basal medium. After replacing the medium with fresh 0.1% BSA medium, the cells are incubated with the test fusion protein of the invention for 3 days. Alamar Blue (Almar Biosciences, Sacramento, Calif.) Is added to each well to a final concentration of 10%. The cells are incubated for 4 hours. Cell viability is measured by reading with a CytoFluor fluorescent reader. For the PGE ₂ assay, human lung fibroblasts are cultured at 5,000 cells / well for 1 day in 96 well plates. After changing the medium to 0.1% BSA basal medium, the cells are incubated with FGF-2 or a fusion protein of the invention for 24 hours with or without IL-1α. The supernatant was collected, and EIA kit (Cayman, Ann Arbor, MI) by assaying for PGE _2. For the IL-6 assay, human lung fibroblasts are cultured at 5,000 cells / well for 1 day in 96 well plates. After changing the medium to 0.1% BSA basal medium, the cells are incubated with FGF-2 for 24 hours with or without the albumin fusion protein of the invention and / or IL-1α. The supernatant is collected and assayed for IL-6 by ELISA kit (Endogen, Cambridge, Mass.).

  Human lung fibroblasts are cultured with FGF-2 or the albumin fusion protein of the present invention in basal medium for 3 days, and then Alamar Blue is added to evaluate the effect on fibroblast proliferation. FGF-2 should exhibit a stimulation of 10-2500 ng / ml that can be used comparable to stimulation with an albumin fusion protein of the invention.

(Cell proliferation based on [3H] thymidine incorporation assay)
The following [3H] thymidine incorporation assay can be used to measure the effect of therapeutic proteins (eg, growth factor proteins) on the growth of cells (eg, fibroblasts, epithelial cells or immature muscle cells).

  Sub-confluent cultures are stopped in the G1 phase by 18 hours incubation in serum-free medium. The therapeutic protein is then added for 24 hours and for the last 4 hours the culture is labeled with [3H] thymidine at a final concentration of 0.33 μM (25 Ci / mmol, Amersham, Arlington Heights, IL). The Incorporated [3H] thymidine is precipitated with ice-cold 10% trichloroacetic acid for 24 hours. The cells are then rinsed continuously with ice cold 10% trichloroacetic acid and then with ice cold water. Following lysis with 0.5M NaOH, the lysate and PBS rinse (500 ml) are pooled and the amount of radioactivity is measured.

(Parkinson model)
The loss of motor function in Parkinson's disease is due to a lack of striatal dopamine resulting from degeneration of nigrostriatal dopaminergic projection neurons. A widely characterized animal model of Parkinson's disease involves systemic administration of 1-methyl-4phenyl 1,2,3,6-tetrahydropyridine (MPTP). In the CNS, MPTP is taken up by astrocytes and catabolized and released by monoamine oxidase B to 1-methyl-4-phenylpyridine (MPP ⁺ ). Subsequently, MPP ⁺ actively accumulates in dopaminergic neurons through dopamine's high affinity reuptake transporter. MPP ⁺ is then concentrated in the mitochondria by an electrochemical gradient and selectively inhibits nicotinamide adenine diphosphate: ubiquinone oxidoreductase (complex I), thereby preventing electron transfer, and Finally, active oxygen is generated.

  It has been demonstrated in a tissue culture paradigm that FGF-2 (basic FGF) has trophic activity on substantia nigra dopaminergic neurons (Ferrari et al., Dev. Biol. 1989). Recently, Dr. Unsikker's group has demonstrated that FGF-2 administration in striatal gel-type implants results in almost complete protection of nigrodopaminergic neurons from toxicity associated with MPTP exposure ( Otto and Unsicker, J. Neuroscience, 1990).

Based on the data using FGF-2, the albumin fusion protein of the present invention has an action similar to that of FGF-2 in enhancing dopaminergic neuron survival in vitro. And albumin fusion proteins of the invention can also test dopaminergic neurons in the striatum in vivo for protection from damage associated with MPTP treatment. The potential effects of the albumin fusion protein of the invention are first tested in vitro in a dopaminergic neuron cell culture paradigm. Cultures are prepared by dissecting the midbrain base plate from Wistar rat embryos on day 14 of gestation. Tissues are separated with trypsin and plated at a density of 200,000 cells / cm ^{2 on} coverslips coated with polyortinin-laminin. The cells are maintained in F12 medium containing Dulbecco's modified Eagle medium and hormone supplement (NI). After 8 days in vitro, cultures are fixed with paraformamide and processed for immunohistochemical staining with tyrosine hydroxylase (a specific marker for dopaminergic neurons). Separate cell cultures are prepared from embryonic rats. The culture medium is changed every 3 days and this factor is also added at each time point.

  Since dopaminergic neurons are isolated from animals at 14 days of gestation (time of development past the stage where dopaminergic progenitor cells proliferate), an increase in the number of tyrosine hydroxylase immunopositive neurons is associated with the survival of dopamine in vitro. An increase in the number of operative neurons is shown. Thus, if the therapeutic protein of the present invention acts to prolong dopaminergic neuron survival, this suggests that this fusion protein may be involved in Parkinson's disease.

(Example 70: Effect of albumin fusion protein of the present invention on proliferation of vascular endothelial cells)
On day 1, human umbilical vein epithelial cells (HUVEC) were supplemented with 4% fetal bovine serum (FBS), 16 units / ml heparin, and 50 units / ml epithelial cell growth supplement (ECGS, Biotechnique, Inc.). Seed in M199 medium containing 2-5 × 10 ⁴ cells / 35 mm dish density. On day 2, the medium was replaced with M199 containing 10% FBS, 8 units / ml heparin. Albumin fusion proteins of the invention and positive controls (eg, VEGF and basal FGF (bFGF) are added at various concentrations. Medium is replaced on days 4 and 6. On day 8, Cell number is determined using a Coulter Counter.

  An increase in the number of HUVECs indicates that the fusion protein can proliferate vascular endothelial cells, while a decrease in the number of HUVECs indicates that the fusion protein inhibits vascular endothelial cells.

(Example 71: Rat corneal wound healing model)
This animal model shows the effect of the albumin fusion polypeptide of the invention on neovascularization. The experimental protocol includes:
Make an incision 1 to 1.5 mm long from the center of the cornea to the stroma.

  Insert a spatula under the lip of the incision facing the cornea outside the eye.

  Make a pocket (its base is 1-1.5 mm from the edge of the eye).

  Place pellets containing 50 ng to 5 μg of the albumin fusion protein of the invention in the pocket.

  Treatment with an albumin fusion protein of the invention can also be applied topically to corneal wounds within a dosage range of 20 mg to 500 mg (daily treatment for 5 days).

Example 72: Diabetic mice and glucocorticoid injured wound healing model
(Diabetes db + / db + mouse model)
In order to demonstrate that the albumin fusion protein of the present invention promotes the healing process, a genetic diabetes mouse model of wound healing is used. The full-thickness wound healing model in db + / db + mice is a well-characterized, clinically relevant and reproducible model of wound wound healing. Healing of diabetic wounds relies on granulation tissue formation and re-epithelialization rather than contraction (Gartner, MH et al., J. Surg. Res. 52: 389 (1992); Greenhalgh, DG et al. Am. J. Pathol. 136: 1235 (1990)).

  This diabetic animal has many of the characteristic characteristics observed in type II diabetes mellitus. Homozygous (db + / db +) mice are obese compared to their normal heterozygous (db + / + m) littermates. Mutant diabetic (db + / db +) mice have a single autosomal recessive mutation (db +) on chromosome 4 (Coleman et al., Proc. Natl. Acad. Sci. USA 77: 283-293 (1982). ). Animals exhibit bulimia, polyposis, and polyuria. Mutant diabetic mice (db + / db +) have elevated blood glucose, increased or normal insulin levels, and suppressed cell-mediated immunity (Mandel et al., J. Immunol. 120: 1375 (1978); Debray -Sachs, M. et al., Clin. Exp. Immunol. 51 (1): 1-7 (1983); Leiter et al., Am. J. of Pathol. 114: 46-55 (1985)). Peripheral neuropathy, myocardial complications, and microvascular injury, basement membrane thickening, and glomerular filtration abnormalities have been described in these animals (Norido, F. et al., Exp. Neurol. 83 (2): 221- Robertson et al., Diabetes 29 (1): 60-67 (1980); Giacomelli et al., Lab Invest. 40 (4): 460-473 (1979); Coleman, DL, Diabetes 31 (Supplement). : 1-6 (1982)). These homozygous diabetic mice develop hyperglycemia, which is resistant to insulin similar to human type II diabetes (Mandel et al., J. Immunol. 120: 1375-1377 (1978). ).

  The features observed in these animals suggest that healing in this model may be similar to that observed in human diabetes (Greenhalgh et al., Am. J. of Pathol. 136: 1235-1246 (1990). ).

  Genetically diabetic female C57BL / KsJ (db + / db +) mice and their non-diabetic (db + / + m) heterozygous littermates are used for this study (Jackson Laboratories). These animals are purchased at 6 weeks of age, and these animals are 8 weeks old at the start of the study. Animals are kept individually and are given food and water ad libitum. All manipulations are performed using aseptic techniques. This experiment is performed according to the rules and guidelines of Human Genome Sciences, Inc. Institutional Animal Care and Use Committee and the Guideline for the Care and Use of Laboratories.

The wound protocol is performed according to previously reported methods (Tsuboi, R. and Rifkin, DB, J. Exp. Med. 172: 245-251 (1990)).
Briefly, on the day of wounding, animals are anesthetized with intraperitoneal injection of Avertin (0.01 mg / mL), 2,2,2-tribromoethanol and 2-methyl-2-butanol dissolved in deionized water. To do. The animal's dorsal area is shaved and the skin is washed with 70% ethanol solution and iodine. The surgical area is dried with sterile gauze before wounding. An 8 mm full-thickness wound is then created using a Keyes tissue punch. Immediately after wounding, the surrounding skin is gently stretched to remove the wound spread. The wound is opened during the experiment. Treatment application is given topically for 5 consecutive days starting from the day of wounding. Prior to treatment, the wound is gently washed with sterile saline and gauze sponge.

  The wound is visually inspected and photographed at a fixed distance on the day of surgery and every two days thereafter. Wound closure is determined by daily measurements on days 1-5 and 8. Wounds are measured horizontally and vertically using a calibrated Jameson caliper. A wound is considered healed when granulation tissue is no longer visible and the wound is covered by a continuous epithelium.

  The albumin fusion protein of the invention is administered in the vehicle for 8 days using different dose ranges (4 mg to 500 mg per wound per day). The vehicle control group received 50 mL of vehicle solution.

  Animals are euthanized on day 8 by intraperitoneal injection of sodium pentobarbital (300 mg / kg). The wound and surrounding skin are then collected for histology and immunohistochemistry. Tissue specimens are placed in 10% neutral buffered formalin in tissue cassettes between biopsy sponges for further processing.

  Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls) are evaluated: 1) vehicle placebo control, 2) untreated group, and 3) treated group.

Wound closure is analyzed by measuring the area on the horizontal and vertical axes and obtaining the total area of the wound. Shrinkage is then assessed by establishing the difference between the initial wound area (day 0) and the post-treatment (day 8) area. This wound area on day 1 is 64 mm ² (size corresponding to the skin punch). The calculation is performed using the following formula:
a. [Open area on day 8]-[Open area on day 1] / [Open area on day 1].

  The specimen is fixed in 10% buffered formalin and the paraffin embedded mass is cut perpendicular to the wound surface (5 mm) and cut using a Reichert-Jung microtome. Routine hematoxylin-eosin (H & E) staining is performed on bisected wound cross sections. Wound histology is used to assess whether the healing process and morphological appearance of the repaired skin has been altered by treatment with the albumin fusion protein of the present invention. This assessment includes verification of the presence of cell accumulation, inflammatory cells, capillaries, fibroblasts, reepithelialization, and epidermal maturation (Greenhalgh, DG et al., Am. J. Pathol. 136: 1235 (1990). ). A calibrated lens micrometer is used by a blinded observer.

  Tissue sections are also immunohistochemically stained with polyclonal rabbit anti-human keratin antibodies using the ABC Elite detection system. Human skin is used as a positive tissue control while nonimmune IgG is used as a negative control. Keratinocyte proliferation is determined by assessing the extent of wound re-epithelialization using a calibrated lens micrometer.

  Proliferating cell nuclear antigen / cyclin (PCNA) in skin specimens is demonstrated by using an anti-PCNA antibody (1:50) using the ABC Elite detection system. Human colon cancer can serve as a positive tissue control, and human brain tissue can be used as a negative tissue control. Each specimen contained sections with primary antibody shedding and replacement with non-immune mouse IgG. The order of these sections is based on the extent of growth on a scale of 0-8 (lower scale reflecting slight growth to higher side reflecting intense growth).

  Experimental data is analyzed using a one-sided t-test. A p value of <0.05 is considered significant.

(Steroid disorder rat model)
Inhibition of wound healing by steroids has been well documented in various in vitro and in vivo systems (Wahl, Glucocorticoids and Wound healing: Anti-Inflammatory Steroid Action: Basic and Clinical Aspects. 280-302 et al. 1989h; J. Immunol. 115: 476-481 (1975); Werb et al., J. Exp. Med.147: 1684-1694 (1978)). Glucocorticoids inhibit angiogenesis, vascular permeability (Ebert et al., An. Inter. Med. 37: 701-705 (1952)), fibroblast proliferation, and collagen synthesis (Beck et al., Growths). Factors.5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61: 703-797 (1978)), as well as causing a transient decrease in circulating monocytes (Haynes). J. Clin. Invest. 61: 703-797 (1978); Wahl, “Glucocorticoids and wound healing”: Antiinflammatory Steroid Action: Basic and Clinical Aspects, Acade. ic Press, New York, delays wound healing by pp 280-302 (1989)). Systemic administration of steroids for impaired wound healing is a well-established phenomenon in rats (Beck et al., Growth Factors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61: 703). Wahl, “Glucocorticoids and wound healing”: Antiinflammation Steroid Action: Basic and Clinical Aspects, Academic Press, New York, pp. 280-A. 86: 2229-2233 (1989)).

  In order to demonstrate that the albumin fusion proteins of the present invention can promote the healing process, a plurality of albumin fusion proteins of the present invention against full thickness excised skin wounds in rats, where healing was impaired by systemic administration of methylprednisolone Evaluate the effect of topical application.

  Young adult male Sprague Dawley rats (weight 250-300 g) (Charles River Laboratories) are used in this experiment. The animals are purchased at 8 weeks of age. The start of the study is 9 weeks old. The healing response of rats is compromised by systemic administration of methylprednisolone (17 mg / kg / rat, intramuscular) at the time of wounding. Animals are raised individually and given food and water ad libitum. All manipulations are performed using aseptic techniques. This study is conducted according to the rules and guidelines of Human Genome Sciences, Inc. Institutional Animal Care and Use Committee and the Guideline for the Care and Use of Laboratory Animals.

  The wound protocol follows above. On the day of wounding, the animals are anesthetized with an intramuscular injection of ketamine (50 mg / kg) and xylazine (5 mg / kg). The animal's dorsal area is shaved and the skin is washed with 70% ethanol and iodine solution. The surgical area is dried with sterile gauze before wounding. An 8 mm full-thickness wound is created using a Keyes tissue punch. The wound is opened during the experiment. Application of the test substance is given topically once a day for 7 consecutive days starting from the day of wounding and then methylmethylprednisolone administration. Prior to treatment, the wound is gently washed with sterile saline and gauze sponge.

  The wound is visually inspected and photographed at a fixed distance on the day of wounding and at the end of treatment. Wound closure is determined by daily measurements on days 1-5 and on day 8. The wound is measured horizontally and vertically using a calibrated Jameson caliper. A wound is considered healed when granulation tissue is no longer visible and the wound is covered by a continuous epithelium.

  The fusion proteins of the invention are administered in the vehicle for 8 days using different dose ranges (4 mg to 500 mg per wound per day). The vehicle control group received 50 mL of vehicle solution.

  Animals are euthanized on day 8 by intraperitoneal injection of sodium pentobarbital (300 mg / kg). The wound and surrounding skin are then collected for histology. Tissue specimens are placed in 10% neutral buffered formalin in tissue cassettes between biopsy sponges for further processing.

  Four groups of 10 animals each (5 with methylprednisolone and 5 without glucocorticoid) are evaluated: 1) untreated group, 2) vehicle placebo control, and 3) treated group.

Wound closure is analyzed by measuring the area on the vertical and horizontal axes and obtaining the total area of the wound. Closure is then assessed by establishing the difference between the initial wound area (day 0) and the post-treatment (day 8) area. This wound area on day 1 is 64 mm ² (size corresponding to the skin punch). The calculation is performed using the following formula:
a. [Open area on day 8]-[Open area on day 1] / [Open area on day 1].

  The specimen is fixed in 10% buffered formalin and the paraffin-embedded mass is cut perpendicular to the wound surface (5 mm) and cut using an Olympus microtome. Routine hematoxylin-eosin (H & E) staining is performed on bisected wound cross sections. The histological examination of the wound makes it possible to assess whether the healing process and morphological appearance of the repaired skin has been improved by treatment with the albumin fusion protein of the invention. A graduated lens micrometer is used by a blinded observer to determine the distance of the wound gap.

  Experimental data is analyzed using a one-sided t-test. A p value of <0.05 is considered significant.

(Example 73: Lymphedema animal model)
The purpose of this experimental approach is to create an appropriate and consistent lymphedema model to test the therapeutic effect of the albumin fusion protein of the present invention on lymphangiogenesis in the rat hind limb and reestablishment of the lymphatic circulation It is to be. Efficacy is measured by swollen volume of the affected limb, quantification of lymphatic vessel volume, total plasma protein volume, and histopathology. Acute lymphedema is observed for 7-10 days. Perhaps more importantly, the chronic progression of edema lasts up to 3-4 weeks.

  Prior to beginning surgery, blood samples are collected for protein concentration analysis. Male rats (body weight approximately 350 g) are dosed with pentobarbital. The right limb is then shaved from the knee to the hip joint. The shaved area is wiped with gauze soaked in 70% EtOH. Blood is drawn for serum total protein testing. After measuring the circumference and volume, mark the two measurement levels (0.5 cm above the heel, midpoint of the back of the foot) and then inject the dye into the foot. 0.05 ml of 1% Evan's Blue is injected into the endothelium of both the right and left footpads. Next, after injection of the dye into the foot, circumference measurement and volume measurement are performed.

  Using the knee joint as a mark, the inguinal groin incision is performed in an annular shape so that the position of the femoral blood vessel can be confirmed. Using forceps and a hemostatic agent, the skin flap is dissected and separated. After confirming the position of the femoral blood vessel, the lymphatic vessel running along the side of the blood vessel and below the blood vessel is located. The main lymphatic vessels in this area are then electrostricted suture ligated.

  Using a microscope, a smooth incision is made in the dorsal muscles of the limb (near the semitendonosis and adductor muscles). The location of the popliteal lymph node is then confirmed. The two proximal and two distal lymph vessels and the distal blood supply of the popliteal node are then ligated with sutures. The popliteal lymph nodes and any associated adipose tissue are then removed by cutting the connective tissue.

  Be careful to manage any mild bleeding that occurs during this procedure. After the lymphatics are occluded, the flap is sealed by using liquid skin (Vetbond (AJ Buck)). Separate skin edges are sealed to the underlying muscle tissue leaving a gap of about 0.5 cm around the leg. The skin may also be secured by suturing to the underlying muscle when necessary.

  In order to avoid infection, animals are housed individually with mesh (no flooring). Recovered animals are checked daily through the optimal edema peak. This peak typically occurs until 5-7 days. The plateau edema peak is then observed. To assess the intensity of lymphedema, the circumference and volume of two designated locations on each limb are measured before surgery and daily for 7 days. The effect of plasma protein on lymphedema is determined and whether protein analysis is a useful test perimeter is also investigated. The weight of both the control and edema limbs is evaluated at two locations. Analysis is performed in a blind manner.

  Circumference measurement: Under short-term gas anesthesia to prevent movement of the limb, the circumference of the limb is measured using a cloth tape measure. Measurements are taken by two different persons at the talus and back of the foot, and then these two readings are averaged. Readings are taken from both control and edema limbs.

  Volume measurement: On the day of surgery, animals are anesthetized with pentobarbital and tested prior to surgery. For daily volumetric measurements, animals are placed under short-term halothane anesthesia (rapid fixation and quick recovery), both legs are shaved, and the legs are marked equally with water resistant markers. The legs are first soaked in water, then each soaked to the marked level and then measured by Buxco edema software (Chen / Victor). The data is recorded by one person, while the other person immerses his leg to the marked area.

  Blood-plasma protein measurement: Blood is removed prior to surgery, centrifuged, and serum separated, and then concluded for total protein and Ca2 + comparison.

  Limb weight comparison: After removing blood, the animals are prepared for tissue collection. The limb is amputated with quillitine, then both experimental and control legs are ligated and amputated and weighed. A second weighing is performed by removing the tibio-cacanal joint and weighing the foot.

  Histological preparation: The transverse muscle located behind the knee (popliteal) region is cut out and placed in a metal mold, filled with freezeGel, soaked in cold methylbutane and placed at −80 ° C. in a labeled sample bag until cut To do. During amputation, muscles are observed for lymphatic vessels under a fluorescence microscope.

(Example 74: Inhibition of TNFα-induced adhesion molecule expression by the albumin fusion protein of the present invention)
The recruitment of lymphocytes to areas of inflammation and angiogenesis relates to specific receptor-ligand interactions between cell surface adhesion molecules (CAM) on lymphocytes and vascular endothelium. This adhesion process is related to intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leukocytes in endothelial cells (EC) in both normal and pathological settings. It follows a multi-step cascade involving adhesion molecule-1 (E-selectin) expression. The expression of these and other molecules in the vascular endothelium determines the efficiency with which leukocytes can adhere to the local vasculature and overflow into the local tissue during the development of an inflammatory response. Local concentrations of cytokines and growth factors are involved in regulating the expression of these CAMs.

  Tumor necrosis factor α (TNF-α) (a potent pro-inflammatory cytokine) is a stimulator of all three CAMs in endothelial cells and can be involved in a wide variety of inflammatory responses, often pathological results Bring.

  The potential of the albumin fusion proteins of the invention to mediate suppression of TNF-α induced CAM expression can be tested. The amount of CAM expression in TNF-α treated protein EC when co-stimulated with a member of the FGF family protein using a modified ELISA assay (which uses EC as a solid phase adsorbent) taking measurement.

To perform this experiment, human umbilical vein endothelial cell (HUVEC) cultures were obtained from pooled cord collections and growth medium (EGM-2; Clonetics, San) supplemented with 10% FCS and 1% penicillin / streptomycin. (Diego, CA) in a humidified incubator at 37 ° C. with 5% CO ₂ . HUVECs are seeded in 96-well plates at a concentration of 1 × 10 ⁴ cells / well in EGM medium at 37 ° C. for 18-24 hours or until confluent. Subsequently, the monolayer was washed 3 times with a serum-free solution of RPMI-1640 supplemented with 100 U / ml penicillin and 100 mg / ml streptomycin, and for 24 hours at 37 ° C. with the specified cytokines and / or growth factors. And processed. After incubation, the cells are then evaluated for CAM expression.

Human umbilical vein endothelial cells (HUVEC) are grown to confluence in standard 96 well plates. Growth medium is removed from the cells and replaced with 90 μl of 199 medium (10% FBS). Samples for testing and positive or negative controls are added to the plate in triplicate (10 μl volume). Plates are incubated at 37 ° C for either 5 hours (selectin and integrin expression) or 24 hours (integrin expression only). The plate is aspirated to remove the media and 100 μl 0.1% paraformaldehyde-PBS (with Ca ⁺⁺ and Mg ⁺⁺ ) is added to each well. Hold the plate at 4 ° C. for 30 minutes.

  The fixative is then removed from the wells and the wells are washed once with PBS (+ Ca, Mg) + 0.5% BSA and drained. Do not let this well dry. 10 μl of diluted primary antibody is added to the test and control wells. Anti-ICAM-1-Biotin, anti-VCAM-1-Biotin and anti-E-selectin-Biotin are used at a concentration of 10 μg / ml (1:10 dilution of 0.1 mg / ml stock antibody). Cells are incubated for 30 minutes at 37 ° C. in a humidified environment. Wells are washed 3 times with PBS (+ Ca, Mg) + 0.5% BSA.

Then 20 μl of diluted ExtrAvidin-Alkaline Phosphatase (1: 5,000 dilution) is added to each well and incubated at 37 ° C. for 30 minutes. Wells are washed 3 times with PBS (+ Ca, Mg) + 0.5% BSA. One tablet of p-nitrophenol phosphate (pNPP) is dissolved in 5 ml of glycine buffer (pH 10.4). 100 μl of pNPP substrate in glycine buffer is added to each test well. Triplicate standard wells were subjected to working dilution of ExtrAvidin-Alkaline Phosphate in glycine buffer (1: 5,000 (10 ⁰ )> 10 ^−0.5 > 10 ⁻¹ > 10 ^−1.5 ). Prepare from 5 μl of each dilution is added to triplicate wells and the resulting AP content in each well is 5.50 ng, 1.74 ng, 0.55 ng, 0.18 ng. 100 μl of pNPP reagent must then be added to each of the standard wells. The plate must be incubated for 4 hours at 37 ° C. A volume of 50 μl of 3M NaOH is added to all wells. The result is quantified at 405 nm in a plate reader. The background subtraction option is used in blank wells filled with glycine buffer only. This template is set to indicate the concentration of AP conjugate in each standard well [5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results are shown as the amount of bound AP conjugate in each sample.

Example 75: Construction of GAS reporter construct
One signaling pathway involved in cell differentiation and proliferation is called the Jaks-STAT pathway. Proteins activated in the Jaks-STAT pathway bind to the gamma activation site “GAS” element or interferon-sensitive response element (“ISRE”) located in the promoters of many genes. Protein binding to these elements alters the expression of related genes.

  The GAS and ISRE elements are recognized by a class of transcription factors called transcriptional signal transducers and transcriptional activators, or “STATs”. There are six members in the STAT family. Stat1 and Stat3 are present in many cell types, similar to Stat2 (so that the response to IFNα is extensive). Stat4 is more limited and is not present in many cell types, but has been found in T helper class I cells after treatment with IL-12. Stat5, originally called breast growth factor, has been found at higher concentrations in other cells, including bone marrow cells. It can be activated in tissue culture cells by many cytokines.

  STATs are activated and translocate from the cytoplasm to the nucleus upon tyrosine phosphorylation by a set of kinases known as the Janus Kinase (“Jaks”) family. Jaks is representative of a unique family of soluble tyrosine kinases and includes Tyk2, Jak1, Jak2, and Jak3. These kinases show significant sequence similarity and are generally catalytically inactive in resting cells.

  Jaks is activated by a wide range of receptors as summarized by the following table. (Modified from review by Schidler and Darnell, Ann. Rev. Biochem. 64: 621-51 (1995)). The cytokine receptor family capable of activating Jaks is divided into two groups: (a) Class 1 is IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL- 11, receptors for IL-12, IL-15, Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF, and thrombopoietin; and (b) class 2 includes IFN-a, IFN-g And IL-10. Class 1 receptors are conserved cysteine motifs (a set of four conserved cysteines and a tryptophan), and a WSXWS motif (membrane proximity region encoding Trp-Ser-Xaa-Trp-Ser (SEQ ID NO: 1113)). Share

  Thus, upon binding of the ligand to the receptor, Jaks is activated, which in turn activates STAT, which then migrates and binds to the GAS element. This entire process is encompassed by the Jaks-STAT signaling pathway. Therefore, activation of the Jaks-STAT pathway reflected by binding of GAS or ISRE elements can be used to indicate proteins involved in cell proliferation and differentiation. For example, growth factors and cytokines are known to activate the Jaks-STAT pathway. (See the table below). Thus, by using a GAS element linked to a reporter molecule, an activator of the Jaks-STAT pathway can be identified.

実施例７８〜８０に記載される生物学的アッセイにおいて使用されるプロモーターエレメントを含む合成ＧＡＳを構築するために、ＰＣＲに基づいたストラテジーを用いてＧＡＳ−ＳＶ４０プロモーター配列を生成する。５’プライマーは、ＩＲＦ１プロモーターにおいて見出され、そして一定の範囲のサイトカインでの誘導の際にＳＴＡＴに結合することが以前に実証されたＧＡＳ結合部位の４つの直列のコピーを含むが（Ｒｏｔｈｍａｎら、Ｉｍｍｕｎｉｔｙ １：４５７−４６８（１９９４））、他のＧＡＳエレメントまたはＩＳＲＥエレメントを代わりに使用し得る。５’プライマーはまた、ＳＶ４０初期プロモーター配列に対して相補的な１８ｂｐの配列も含み、そしてＸｈｏＩ部位に隣接する。５’プライマーの配列は以下である：

To construct a synthetic GAS containing the promoter elements used in the biological assays described in Examples 78-80, a GAS-SV40 promoter sequence is generated using a PCR-based strategy. The 5 ′ primer contains four tandem copies of the GAS binding site found in the IRF1 promoter and previously demonstrated to bind to STAT upon induction with a range of cytokines (Rothman et al. , Immunity 1: 457-468 (1994)), other GAS elements or ISRE elements may be used instead. The 5 'primer also contains an 18 bp sequence complementary to the SV40 early promoter sequence and is flanked by XhoI sites. The sequence of the 5 ′ primer is:

下流プライマーはＳＶ４０プロモーターに対して相補的であり、そしてＨｉｎｄ ＩＩＩ部位に隣接する：５’：ＧＣＧＧＣＡＡＧＣＴＴＴＴＴＧＣＡＡＡＧＣＣＴＡＧＧＣ：３’（配列番号１１１５）。

The downstream primer is complementary to the SV40 promoter and flanks the Hind III site: 5 ′: GCGGCAAGCTTTTTGCAAAGCCTAGGC: 3 ′ (SEQ ID NO: 1115).

  PCR amplification is performed using the SV40 promoter template present in the B-gal: promoter plasmid obtained from Clontech. The resulting PCR fragment is digested with XhoI / Hind III and subcloned into BLSK2- (Stratagene). Sequencing with forward and reverse primers confirms that the insert contains the following sequence:

このＧＡＳプロモーターエレメントがＳＶ４０プロモーターに結合されると、次に、ＧＡＳ：ＳＥＡＰ２レポーター構築物を操作する。ここで、レポーター分子は、分泌アルカリホスファターゼ、すなわち「ＳＥＡＰ」である。しかし、明らかに、この実施例または他の実施例のいずれにおいても、任意のレポーター分子が、ＳＥＡＰの代わりとなり得る。ＳＥＡＰの代わりに使用され得る周知のレポーター分子としては、クロラムフェニコールアセチルトランスフェラーゼ（ＣＡＴ）、ルシフェラーゼ、アルカリホスファターゼ、β−ガラクトシダーゼ、グリーン蛍光タンパク質（ＧＦＰ）、または抗体により検出可能な任意のタンパク質が挙げられる。

Once this GAS promoter element is bound to the SV40 promoter, the GAS: SEAP2 reporter construct is then manipulated. Here, the reporter molecule is secreted alkaline phosphatase, or “SEAP”. Clearly, however, any reporter molecule in this or other examples can replace SEAP. Well known reporter molecules that can be used in place of SEAP include chloramphenicol acetyltransferase (CAT), luciferase, alkaline phosphatase, β-galactosidase, green fluorescent protein (GFP), or any protein detectable by an antibody. Can be mentioned.

  From the Clontech, the synthetic GAS-SV40 promoter element identified by the above sequence was effectively replaced with the amplified GAS: SV40 promoter element using HindIII and XhoI to create a GAS-SEAP vector. Subcloning into the obtained pSEAP-promoter vector. However, this vector does not contain a neomycin resistance gene and is therefore not preferred for mammalian expression systems.

  Thus, to create a mammalian stable cell line that expresses the GAS-SEAP reporter, the GAS-SEAP cassette is removed from the GAS-SEAP vector using SalI and NotI and the GAS-SEAP / Neo vector is generated. To do this, these restriction sites in the multicloning site are used to insert into a backbone vector containing a neomycin resistance gene (eg, pGFP-1 (Clontech)). Once the vector is transfected into mammalian cells, the vector can then be used as a reporter molecule for GAS binding as described in Examples 78-80.

  Other constructs can be made using the above description and replacing the GAS with a different promoter sequence. For example, the construction of a reporter molecule comprising NF-KB and EGR promoter sequences is described in Examples 78-82. However, many other promoters can be replaced using the protocols described in these examples. For example, SRE, IL-2, NFAT, or osteocalcin promoter alone or in combination (eg, GAS / NF-KB / EGR, GAS / NF-KB, Il-2 / NFAT, or NF-KB / GAS ) Can be substituted. Similarly, other cell lines (eg, HELA (epithelial cells), HUVEC (endothelial cells), Reh (B cells), Saos-2 (osteoblasts), HUVAC (aortic cells), or cardiomyocytes) can be used as reporters. It can be used to test the activity of the construct.

Example 76: Assay for SEAP activity
As a reporter molecule for the assays described in the Examples disclosed herein, SEAP activity is assayed using Tropix Phospho-light Kit (Catalog Number BP-400) according to the following general procedure . Tropix Phospho-light Kit supplies dilution buffer, assay buffer, and reaction buffer used in the following.

  Fill the dispenser with 2.5 × dilution buffer and dispense 15 μl of 2.5 × dilution buffer to an Optiplate containing 35 μl of the supernatant containing the albumin fusion protein of the present invention. Seal the plate with a plastic sealer and incubate at 65 ° C. for 30 minutes. Keep the Optiplate apart to avoid uneven heating.

  Cool the sample to room temperature for 15 minutes. Empty the dispenser and fill with assay buffer. Add 50 ml assay buffer and incubate at room temperature for 5 minutes. Empty the dispenser and fill the reaction buffer (see table below). Add 50 μl of reaction buffer and incubate for 20 minutes at room temperature. The intensity of the chemiluminescent signal is time dependent, and it takes about 10 minutes to read 5 plates with a luminometer, so process 5 plates at a time and start the second set after 10 minutes Should do.

  Read the relative light unit in the luminometer. Set H12 as blank and print the result. An increase in chemiluminescence indicates reporter activity.

（実施例７７：ニューロン活性を同定するアッセイ）
細胞が分化および増殖を経る場合、一群の遺伝子が多くの異なるシグナル伝達経路を介して活性化される。これらの遺伝子の１つであるＥＧＲ１（初期増殖応答遺伝子１）は、活性化時に種々の組織および細胞型において誘導される。ＥＧＲ１のプロモーターはこのような誘導を担う。レポーター分子に結合したＥＧＲ１プロモーターを使用して、細胞を活性化する本発明のアルブミン融合タンパク質の能力を評価し得る。

Example 77: Assay to identify neuronal activity
As cells undergo differentiation and proliferation, a group of genes is activated through many different signaling pathways. One of these genes, EGR1 (early growth response gene 1), is induced in various tissues and cell types upon activation. The EGR1 promoter is responsible for such induction. The EGR1 promoter coupled to a reporter molecule can be used to assess the ability of the albumin fusion proteins of the present invention to activate cells.

  Specifically, the following protocol is used to assess neuronal activity in the PC12 cell line. PC12 cells (rat pheochromocytoma cells) are activated by many mitogens such as TPA (tetradecanoylphorbol acetate), NGF (nerve growth factor), and EGF (epidermal growth factor). It is known to proliferate and / or differentiate. During this treatment, EGR1 gene expression is activated. Thus, PC12 cell activation by the albumin fusion protein of the invention can be assessed by stably transfecting PC12 cells with a construct comprising an EGR promoter linked to a SEAP reporter.

The EGR / SEAP reporter construct can be assembled by the following protocol. The EGR-1 promoter sequence (-633- + 1) (Sakamoto K et al., Oncogene 6: 867-871 (1991)) can be PCR amplified from human genomic DNA using the following primers:
First primer: 5′GCCGCTCGAGGGATGACACGCGATAGAACCCCGG-3 ′ (SEQ ID NO: 1117)
Second primer: 5 ′ GCGAAGCTTCGCGCACTCCCCGGATCCGCTCTC-3 ′ (SEQ ID NO: 1118).

  The EGR1 amplification product can then be inserted into this vector using the GAS: SEAP / Neo vector created in Example 75. The GAS: SEAP / Neo vector is linearized using the restriction enzymes XhoI / HindIII and the GAS / SV40 stuffer is removed. The same enzyme is used to restrict the EGR1 amplification product. Ligate the vector and the EGR1 promoter.

  To prepare a 96 well plate for cell culture, a coating solution (1:30 dilution of collagen type I (Upstate Biotech Inc. Cat. No. 08-115) with 30% ethanol (sterile filtration)) in one 10 cm. Add 2 ml per plate, or 50 ml per well of a 96 well plate and allow to air dry for 2 hours.

  PC12 cells were pre-coated on 10 cm tissue culture dishes supplemented with penicillin (100 units / ml) and streptomycin (100 μg / ml), 10% horse serum (JRH BIOSCIENCES, catalog number 12449-78P), 5% heat Conventionally grown in RPMI-1640 medium (Bio Whittaker) with inactivated fetal bovine serum (FBS). Split from 1 to 4 every 3-4 days. Cells are removed from the plate by scraping and resuspended by pipetting up and down more than 15 times.

  The EGR / SEAP / Neo construct is transfected into PC12 using techniques known in the art. EGR-SEAP / PC12 stable cells are obtained by growing the cells in 300 μg / ml G418. Medium without G418 is used for routine growth, but cells should be regrown in 300 μg / ml G418 for 2 passages every 1-2 months.

  To assay for neuronal activity, 10 cm plates with cells at approximately 70-80% confluence are screened by removing old media. Cells are washed once with PBS (phosphate buffered saline). Cells are then starved overnight in low serum medium (RPMI-1640 with 1% horse serum and 0.5% FBS with antibiotics).

The next morning, remove the media and wash the cells with PBS. Scrape the cells from the plate and suspend the cells in 2 ml of low serum medium. The cell number is counted and lower serum medium is added to reach a final cell density of 5 × 10 ⁵ cells / ml.

200 μl of cell suspension is added to each well of a 96 well plate (equivalent to 1 × 10 ⁵ cells / well). A series of different concentrations of the albumin fusion protein of the invention are added at 37 ° C. for 48-72 hours. As a positive control, a growth factor known to activate PC12 cells via EGR (eg, 50 ng / μl nerve growth factor (NGF)) can be used. Typically, more than 50-fold SEAP induction is seen in positive control wells. SEAP assays can be routinely performed using techniques known in the art and / or as described in Example 76.

Example 78: Assay for T cell activity
The following protocol is used to assess T cell activity by identifying factors and determining whether the albumin fusion proteins of the present invention proliferate and / or differentiate T cells. T cell activity is assessed using the GAS / SEAP / Neo construct generated in Example 75. Thus, factors that increase SEAP activity indicate the ability to activate the Jaks-STATS signaling pathway. The T cells used in this assay are Jurkat T cells (ATCC accession number TIB-152), but Molt-3 cells (ATCC accession number CRL-1552) and Molt-4 cells (ATCC accession number CRL-1582) cells. Can also be used.

  Jurkat T cells are lymphoblastic CD4 + Th1 helper cells. To generate a stable cell line, approximately 2 million Jurkat cells are transfected with GAS-SEAP / neo vectors using DMRIE-C (Life Technologies) (transfection procedure described below). Transfected cells are seeded at a density of approximately 20,000 cells / well and transfectants that are resistant to 1 mg / ml geneticin are selected. Resistant colonies are grown and then tested for their response to increasing concentrations of interferon gamma. The dose response of selected clones is shown.

  Specifically, the following protocol provides sufficient cells for 75 wells containing 200 μl of cells. Therefore, to produce sufficient cells for multiple 96-well plates, this is either scaled up or performed in multiple. Jurkat cells are maintained in RPMI + 10% serum and 1% Pen-Strep. Combine 2.5 ml of OPTI-MEM (Life Technologies) and 10 μg of plasmid DNA in a T25 flask. Add 2.5 ml OPTI-MEM containing 50 μl DMRIE-C and incubate at room temperature for 15-45 minutes.

During the incubation period, the cell concentration is counted, the required number of cells (10 ⁷ per transfection) is spun down and resuspended in OPTI-MEM to a final concentration of 10 ⁷ cells / ml. . Then 1 ml of 1 × 10 ⁷ cells in OPTI-MEM is added to the T25 flask and incubated at 37 ° C. for 6 hours. After incubation, 10 ml RPMI + 15% serum is added.

  The Jurkat: GAS-SEAP stable reporter strain is maintained in RPMI + 10% serum, 1 mg / ml geneticin, and 1% Pen-Strep. These cells are treated with one or more various concentrations of the fusion protein of the present invention.

  Cells should be washed on the day of treatment with the fusion protein and resuspended in fresh RPMI + 10% serum to a density of 500,000 cells per ml. The exact number of cells required depends on the number of fusion proteins screened and the number of different concentrations of the fusion protein. For one 96-well plate, approximately 10 million cells (100 million cells for 10 plates) are required.

  Well dishes containing Jurkat cells treated with the fusion protein are placed in an incubator for 48 hours (Note: this time can vary between 48-72 hours). Then 35 μl of sample from each well is transferred to an opaque 96 well plate using a 12 channel pipette. The opaque plate should be covered (using a cellophane cover) and stored at −20 ° C. until the SEAP assay is performed according to Example 76. Plates containing the remaining treated cells are placed at 4 ° C. and serve as a source of material to repeat the assay on specific wells, if desired.

  As a positive control, 100 units / ml interferon γ can be used, which is known to activate Jurkat T cells. Typically, more than 30-fold induction is observed in positive control wells.

  The protocol described above can be used to generate both transient and stable transfected cells, as will be apparent to those skilled in the art.

Example 79: Assay for T cell activity
NF-KB (nuclear factor KB) is produced by exposure to LPS or thrombin by a wide variety of drugs, including inflammatory cytokines IL-1 and TNF, CD30 and CD40, lymphotoxin-α and lymphotoxin-β. As well as transcription factors activated by the expression of specific viral gene products. As a transcription factor, NF-KB expresses genes involved in immune cell activation, controls apoptosis (NF-KB appears to protect cells from apoptosis), B and T cell development, antiviral Modulates response and antimicrobial response, as well as multiple stress responses.

  In unstimulated conditions, NF-KB is retained in the cytoplasm with I-KB (inhibitor KB). However, upon stimulation, I-KB is phosphorylated and degraded, causing NF-KB to shuttle to the nucleus, thereby activating transcription of the target gene. Examples of target genes activated by NF-KB include IL-2, IL-6, GM-CSF, ICAM-1 and class 1 MHC.

  Due to its central role and ability to respond to a range of stimuli, reporter constructs that utilize the NF-KB promoter element are used to screen fusion proteins. NF-KB activators or inhibitors are useful in the treatment, prevention and / or diagnosis of diseases. For example, inhibitors of NF-KB may be used to treat diseases associated with acute or chronic NF-KB activation (eg, rheumatoid arthritis).

A PCR based strategy is used to construct a vector containing the NF-KB promoter element. The upstream primer contains four tandem copies of the NF-KB binding site (GGGGACTTTCCCC) (SEQ ID NO: 1119), an 18 bp sequence complementary to the 5 ′ end of the SV40 early promoter sequence, and adjacent to the XhoI site To:
5 ′: GCGGCCTCGAGGGGACTTTCCCCGGGGACTTTCCGGGGACTTTCCGGACTTCCATCCTGCCATCTCAATTTAG: 3 ′ (SEQ ID NO: 1120).

The downstream primer is complementary to the 3 ′ end of the SV40 promoter and is adjacent to the HindIII site:
5 ′: GCGGCAAGCTTTTTGCAAAGCCTAGGC: 3 ′ (SEQ ID NO: 1115).

  PCR amplification is performed using the SV40 promoter template present in the pB-gal: promoter plasmid obtained from Clontech. The resulting PCR fragment is digested with XhoI and HindIII and subcloned into BLSK2- (Stratagene). Sequencing using T7 and T3 primers confirms that the insert contains the following sequence:

次に、ＸｈｏＩおよびＨｉｎｄＩＩＩを使用して、ｐＳＥＡＰ２−プロモータープラスミド（Ｃｌｏｎｔｅｃｈ）に存在するＳＶ４０最小プロモーターエレメントをこのＮＦ−ＫＢ／ＳＶ４０フラグメントで置換する。しかし、このベクターはネオマイシン耐性遺伝子を含まず、そしてそれゆえ哺乳動物の発現系には好ましくない。

XhoI and HindIII are then used to replace the SV40 minimal promoter element present in the pSEAP2-promoter plasmid (Clontech) with this NF-KB / SV40 fragment. However, this vector does not contain a neomycin resistance gene and is therefore not preferred for mammalian expression systems.

  To create a stable mammalian cell line, the NF-KB / SV40 / SEAP cassette is removed from the above NF-KB / SEAP vector using the restriction enzymes SalI and NotI and inserted into a vector containing neomycin resistance. . Specifically, after pGFP-1 was restricted with SalI and NotI, the NF-KB / SV40 / SEAP cassette was inserted into pGFP-1 (Clontech) to replace the GFP gene.

  Once the NF-KB / SV40 / SEAP / Neo vector is generated, stable Jurkat T cells are generated and maintained according to the protocol described in Example 76. Similarly, a method for assaying fusion proteins comprising these stable Jurkat T cells is also described in Example 76. As a positive control, exogenous TNFα (0.1, 1, 10 ng) is added to wells H9, H10, and H11, typically 5-10 fold activation is observed.

Example 80: Assay to identify myeloid activity
The following protocol is used to assess the myeloid activity of an albumin fusion protein of the present invention by determining whether the fusion protein expands and / or differentiates myeloid cells. Myeloid cell activity is assessed using the GAS / SEAP / Neo construct produced in Example 75. Thus, factors that increase SEAP activity exhibit the ability to activate the Jaks-STATS signaling pathway. The myeloid cells used in this assay are U937 (which is a pre-monocyte cell line), but TF-1, HL60, or KG1 can also be used.

To transiently transfect U937 cells with the GAS / SEAP / Neo construct produced in Example 75, the DEAE-Dextran method (Kharbanda et al., 1994, Cell Growth & Differentiation, 5: 259-265). Is used. First, 2 × 10 ⁷ U937 cells are collected and washed with PBS. U937 cells are usually grown in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) supplemented with 100 units / ml penicillin and 100 mg / ml streptomycin.

Next, 0.5mg / ml DEAE-Dextran, GAS-SEAP2 plasmid DNA 8μg, 140mM NaCl, 5mM KCl, 375μM Na 2 HPO 4 · 7H 2 O, 20mM Tris of 1ml containing 1 mM MgCl _2, and 675μM CaCl ₂ Suspend cells in HCl (pH 7.4) buffer. Incubate at 37 ° C for 45 minutes.

  Cells are washed with RPMI 1640 medium containing 10% FBS, then resuspended in 10 ml complete medium and incubated at 37 ° C. for 36 hours.

  GAS-SEAP / U937 stable cells are obtained by growing the cells in 400 μg / ml G418. Although medium without G418 is used for routine growth, cells should be regrown in 400 μg / ml G418 every 2 to 2 months.

These cells are tested by collecting 1 × 10 ⁸ cells (which is sufficient for 10 96-well plate assays) and washed with PBS. Suspend cells in the above 200 ml growth medium at a final density of 5 × 10 ⁵ cells / ml. Plate 200 μl of cells per well (ie, 1 × 10 ⁵ cells / well) in a 96 well plate.

  Add different concentrations of fusion protein. Incubate at 37 ° C for 48-72 hours. As a positive control, 100 units / ml of interferon γ can be used, which is known to activate U937 cells. Typically, over 30-fold induction is observed in positive control wells. The supernatant is SEAP assayed according to methods known in the art and / or the protocol described in Example 76.

Example 81: Assay to identify changes in small molecule concentration and membrane permeability
Binding of the ligand to the receptor is known to alter intracellular levels of small molecules such as calcium, potassium, sodium and pH, as well as membrane potential. These changes can be measured in assays to identify fusion proteins that bind to specific cellular receptors. The following protocol describes an assay for calcium, but this protocol is easily modified to detect changes in potassium, sodium, pH, membrane potential, or any other small molecule detectable by a fluorescent probe. Can be done.

  The following assay uses a Fluorometric Imaging Plate Reader (“FLIPR”) to measure changes in fluorescent molecules (Molecular Probes) that bind to small molecules. Obviously, any fluorescent molecule that detects small molecules can be used in place of the calcium fluorescent molecule, fluo-4 (Molecular Probe, Inc .; catalog number: F-14202) used herein.

For adherent cells, seed cells at 10,000-20,000 cells / well in Co-star black 96 well plates with clear bottom. Incubate the plate in a CO ₂ incubator for 20 hours. Adherent cells are washed twice with 200 μl HBSS (Hanks balanced salt solution) in a Biotek washer, leaving 100 μl buffer after the last wash.

A stock solution of 1 mg / ml fluo-4 is made with 10% pluronic acid DMSO. To load the cells with fluo-4, 12 μg / ml fluo-4 (50 μl) is added to each well. The plate is incubated for 60 minutes at 37 ° C. in a CO ₂ incubator. The plate is washed 4 times with HBSS in a Biotek washer leaving 100 μl of buffer.

For non-adherent cells, the cells are spun down from the culture medium. Cells are resuspended to 2-5 × 10 ⁶ cells / ml with HBSS in a 50 ml conical tube. For each ml of cell suspension, add 4 μl of a 1 mg / ml fluo-4 solution in 10% pluronic acid DMSO. The tube is then placed in a 37 ° C. water bath for 30-60 minutes. Cells are washed twice with HBSS, resuspended to 1 × 10 ⁶ cells / ml and dispensed into microplates at 100 μl / well. Centrifuge the plate at 1000 rpm for 5 minutes. The plate is then washed once in Denley Cell Wash with 200 μl, and the final volume is made 100 μl by an aspiration process.

  For non-cell based assays, each well contains a fluorescent molecule such as fluo-4. Fusion protein is added to the wells and changes in fluorescence are detected.

To measure intracellular calcium fluorescence, the FLIPR is set for the following parameters: (1) System gain is 300-800 mW; (2) Exposure time is 0.4 seconds (3) Camera F / Stop is F / 2; (4) Excitation is 488 nm; (5) Emission is 530 nm; and (6) Sample addition is 50 μl. An increase in luminescence at 530 nm indicates an extracellular signaling event attributed to either the albumin fusion protein of the invention or a molecule induced by the albumin fusion protein of the invention, which is intracellular Ca ^++. This causes an increase in concentration.

Example 82: Assay to identify tyrosine kinase activity
Protein tyrosine kinases (PTKs) represent a diverse group of transmembrane and cytoplasmic kinases. Within the receptor protein tyrosine kinase (RPTK) group are a range of mitogenic and metabolic growth factor receptors, including PDGF, FGF, EGF, NGF, HGF and the insulin receptor subfamily. In addition, there is a large family of RPTKs whose corresponding ligands are unknown. RPTK ligands include primarily secreted low molecular weight proteins, but also include membrane-bound and extracellular matrix proteins.

  Activation of RPTK by ligand involves ligand-mediated receptor dimerization, which results in transphosphorylation of receptor subunits and activation of cytoplasmic tyrosine kinases. Cytoplasmic tyrosine kinases include receptor-related tyrosine kinases of the src family (eg, src, yes, lck, lyn, fyn) and non-receptor-bound and cytosolic protein tyrosine kinases such as the Jak family. These members mediate signaling induced by the cytokine superfamily of receptors (eg, interleukins, interferons, GM-CSF and leptin).

  Since there are a wide variety of known factors that can stimulate tyrosine kinase activity, it is identified whether the albumin fusion protein of the present invention or a molecule induced by the albumin fusion protein of the present invention can activate the tyrosine kinase signaling pathway Interesting to do. Accordingly, the following protocol is designed to identify such molecules that can activate the tyrosine kinase signaling pathway.

  Target cells (eg, primary keratinocytes) are seeded at a density of approximately 25,000 cells / well in a 96-well Loprodyne Silent Screen Plate purchased from Nalge Nunc (Naperville, IL). Plates are sterilized by rinsing twice with 100% ethanol for 30 minutes, rinsed with water, and dried overnight. Several plates can be purchased from 100 ml of cell culture grade type I collagen (50 mg / ml), gelatin (2%) or polylysine (50 mg / ml), all of which are from Sigma Chemicals (St. Louis, MO) ) Or coated with 10% Matrigel purchased from Becton Dickinson (Bedford, Mass.) Or calf serum for 2 hours, rinsed with PBS, and stored at 4 ° C. Growth medium was seeded at 5,000 cells / well and the manufacturer Alamar Biosciences, Inc. Cell proliferation on these plates is assayed by indirect quantification of cells after 48 hours using alamarBlue as described by (Sacramento, CA). Cover the Loprodyne Silent Screen Plate using Falcon plate cover # 3071 from Becton Dickinson (Bedford, MA). Falcon Microtest III cell culture plates can also be used in some proliferation experiments.

To prepare the extract, A431 cells are seeded on a nylon membrane of Loprodyne plates (20,000 / 200 ml / well) and cultured overnight in complete medium. Cells are allowed to rest by incubating in serum-free basal medium for 24 hours. After treatment with EGF (60 ng / ml) or different concentrations of the albumin fusion protein of the invention for 5-20 minutes, the medium is removed and 100 ml of extraction buffer (20 mM HEPES pH 7.5, 0.15 M NaCl, 1% Triton X-100, 0.1% SDS, 2 mM Na ₃ VO ₄ , 2 mM Na ₄ P ₂ O ₇ and a cocktail of protease inhibitors obtained from Boehringer Mannheim (Indianapolis, Ind.)) Were added to each well, The plate is then shaken on a rotary shaker at 4 ° C. for 5 minutes. The plate is then placed in a vacuum transfer manifold and the extract is filtered through a 0.45 mm membrane at the bottom of each well using house vacuum. The extract is collected in a 96 well capture / assay plate at the bottom of the vacuum manifold and immediately placed on ice. To obtain a clarified extract by centrifugation, solubilize with detergent for 5 minutes, then remove the contents of each well and centrifuge at 16,000 xg for 15 minutes at 4 ° C.

  The filtered extract is tested for the level of tyrosine kinase activity. A number of methods for detecting tyrosine kinase activity are known, but one method is described herein.

  In general, the tyrosine kinase activity of the albumin fusion proteins of the invention is assessed by determining the ability to phosphorylate tyrosine residues on a particular substrate (biotinylated peptide). Biotinylated peptides that can be used for this purpose include PSK1 (corresponding to amino acids 6-20 of the cell division kinase cdc2-p34) and PSK2 (corresponding to amino acids 1-17 of gastrin). Both peptides are a range of substrates for tyrosine kinases and are available from Boehringer Mannheim.

The tyrosine kinase reaction is set up by adding the following components in order. First, 10 μl of 5 μM biotinylated peptide was added, then 10 μl ATP / Mg ₂₊ (5 mM ATP / 50 mM MgCl ₂ ), then 10 μl 5 × assay buffer (40 mM imidazole hydrochloride, pH 7.3, 40 mM β-glyceroline). Acid salt, 1 mM EGTA, 100 mM MgCl ₂ , 5 mM MnCl ₂ , 0.5 mg / ml BSA), then 5 μl sodium vanadate (1 mM) and then 5 μl water are added. The components are mixed gently and the reaction mixture is preincubated at 30 ° C. for 2 minutes. Control enzyme or filtered supernatant (10 μl) is added to initiate the reaction.

  The tyrosine kinase assay reaction is then stopped by adding 120 mM EDTA (10 μl) and the reaction is placed on ice.

  Tyrosine kinase activity is determined by transferring a 50 μl aliquot of the reaction mixture to a microtiter plate (MTP) module and incubating at 37 ° C. for 20 minutes. This associates the streptavidin-coated 96 well plate with the biotinylated peptide. Wash the MTP module 4 times with 300 μl / well PBS. Next, 75 μl of anti-phosphotyrosine antibody conjugated to horseradish peroxidase (anti-P-Tyr-POD (0.5 u / ml)) is added to each well and incubated at 37 ° C. for 1 hour. . The wells are washed as above.

  Next, 100 μl of peroxidase substrate solution (Boehringer Mannheim) is added and incubated at room temperature for at least 5 minutes (up to 30 minutes). The absorbance of the sample at 405 nm is measured using an ELISA reader. The level of bound peroxidase activity is quantified using an ELISA reader. This reflects the level of tyrosine kinase activity.

Example 83: Assay to identify phosphorylation activity
As a possible alternative and / or complement of the protein tyrosine kinase activity assay described in Example 82, an assay that detects activation (phosphorylation) of key intracellular signaling intermediates is also used. Can do. For example, as described below, one particular assay can detect tyrosine phosphorylation of Erk-1 and Erk-2 kinases. However, Raf, JNK, p38 MAP, Map kinase kinase (MEK), MEK kinase, Src, muscle specific kinase (MuSK), other molecules such as IRAK, Tec and Janus, and any other phosphoserine, phosphotyrosine or Phosphorylation of phosphothreonine molecules can be detected by substituting Erk-1 or Erk-2 with these molecules in the following assay.

  Specifically, the wells of a 96-well ELISA plate are coated with 0.1 ml protein G (1 μg / ml) for 2 hours at room temperature (RT) to produce an assay plate. The plates are then rinsed with PBS and blocked with 3% BSA / PBS for 1 hour at RT. Protein G plates are then treated with two commercially available monoclonal antibodies against Erk-1 and Erk-2 (100 ng / well) (Santa Cruz Biotechnology) (1 hour at RT). (To detect other molecules, this process can be easily modified by exchanging monoclonal antibodies that detect any of the above molecules). After rinsing 3-5 times with PBS, the plates are stored at 4 ° C. until use.

  A431 cells are seeded at 20,000 / well in 96-well Loprodyne filter plates and cultured overnight in growth medium. The cells are then starved in basal medium (DMEM) for 48 hours and then treated with EGF (6 ng / well) or various concentrations of the fusion protein of the invention for 5-20 minutes. The cells are then solubilized and the extract is filtered and placed directly into the assay plate.

  After incubating with the extract for 1 hour at RT, the wells are rinsed again. As a positive control, a commercially available MAP kinase preparation (10 ng / well) is used instead of the A431 extract. The plates are then treated with a commercially available polyclonal (rabbit) antibody (1 μg / ml) that specifically recognizes phosphorylated epitopes of Erk-1 and Erk-2 kinases (1 hour at RT). This antibody is biotinylated by standard procedures. The bound polyclonal antibody is then quantified in a Wallac DELFIA instrument by sequentially incubating with europium streptavidin and europium fluorescence enhancer (time-resolved fluorescence). An increase in fluorescent signal over background indicates a molecule that is phosphorylated by the fusion protein of the invention or induced by the albumin fusion protein of the invention.

Example 84: Assay for stimulation of bone marrow CD34 + cell proliferation
This assay is based on the ability of human CD34 + to grow in the presence of hematopoietic growth factors and evaluates the ability of the fusion proteins of the invention to stimulate the growth of CD34 + cells.

  The most mature precursor has previously been shown to respond to only a single signal. More immature precursors require at least two signals to respond. Thus, to test the effect of the fusion protein of the invention on the hematopoietic activity of a wide range of ancestral cells, the assay includes a given fusion protein of the invention in the presence or absence of hematopoietic growth factors. The isolated cells are cultured for 5 days in the presence of stem cell factor (SCF) in combination with the test sample. SCF alone has a very limited effect on bone marrow (BM) cell proliferation and acts only under these conditions as a “survival” factor. However, when combined with any factor that exhibits a stimulatory effect on these cells (eg, IL-3), SCF produces a synergistic effect. Thus, such activity can be readily detected if the tested fusion protein has a stimulatory effect on hematopoietic ancestry. Normal BM cells do not appear to detect any inhibitory effects of a given fusion protein because they have a low level of cycling cell. Accordingly, assays for inhibitory effects on ancestors are preferably tested in cells that are first subjected to in vitro stimulation with SCF + IL + 3 and then contacted with a compound that is evaluated for inhibition of such induced proliferation. .

Briefly, CD34 + cells are isolated using methods known in the art. These cells are thawed and resuspended in medium (QBSF 60 serum-free medium with 1% L-glutamine (500 ml) (Quality Biological Inc., Gaithersburg, MD Cat # 160-204-101)). . After some gentle centrifugation at 200 xg, the cells are allowed to sit for 1 hour. The cell number is adjusted to 2.5 × 10 ⁵ cells / ml. During this time, 100 μl of sterile water is added to the peripheral wells of a 96 well plate. In this assay, the only cytokines that can be tested with the albumin fusion protein of the invention are 50 ng / ml rhSCF (R & D Systems, Minneapolis, Minn., Cat # 255-SC) and 30 ng / ml rhSCF and rhIL-3. (R & D Systems, Minneapolis, MN, Cat # 203-ML). After 1 hour, 10 μl of the prepared cytokine, various concentrations of the albumin fusion protein of the invention and 20 μl of diluted cells are added to the medium, which already has been added to a final total volume of 100 μl. Present in. The plate is then placed in a 37 ° C / 5% CO ₂ incubator for 5 days.

  18 hours before the assay, 0.5 μCi / well of [3H] thymidine is added to each well in a volume of 10 μl to determine the growth rate. This experiment is terminated by collecting cells from each 96 well plate into a filter mat using a Tomtec Harvester 96. After collection, the filter mat is dried, trimmed, and placed in an Omnifilter assembly consisting of one Omnifilter plate and one OmniFilter tray. 60 μl of Microscint is added to each well and the plate is sealed with TopSeal-A press-on feeling film. A bar code 15 sticker is secured to the first plate for counting. The sealed plate is then filled and the level of radioactivity is determined via the Packard Top Count and the printed data is collected for analysis. The radioactivity level reflects the amount of cell proliferation.

  The study described in this example tests the activity of a given fusion protein that stimulates bone marrow CD34 + cell proliferation. One skilled in the art can readily modify the illustrated examples to test the activity of the albumin fusion proteins and polynucleotides (eg, gene therapy) of the present invention and their agonists and antagonists. The ability of the albumin fusion protein of the invention to stimulate the proliferation of bone marrow CD34 + cells is useful for the diagnosis and treatment of disorders in which the albumin fusion protein and / or polynucleotide corresponding to this fusion protein affects the immune system and hematopoiesis. It shows that. Exemplary uses are described in the “Immune Activity” and “Infectious Diseases” sections above, and elsewhere in this specification.

  Example 85: Assay for Extracellular Matrix Enhanced Cell Response (EMCCR) The purpose of the Extracellular Matrix Enhanced Cell Response (EMECR) assay is to act on hematopoietic stem cells in relation to extracellular matrix (ECM) induced signals. It is to evaluate the ability of the fusion protein of the present invention.

In response to signals received from the surrounding microenvironment, cells respond to regulatory factors. For example, fibroblasts, as well as endothelial and epithelial stem cells cannot replicate in the absence of signals from the ECM. Hematopoietic stem cells can undergo self-renewal in the bone marrow but do not undergo self-renewal in in vitro suspension culture. The ability of stem cells to undergo self-renewal in vitro depends on their interaction with stromal cells and the ECM protein fibronectin (fn). Adsorption of cells to fn is α ₅ . β ₁ and α ₄ . Mediated by β ₁ integrin receptors, these receptors are expressed by human and mouse hematopoietic stem cells. The factors that integrate in the ECM environment and are responsible for stimulating stem cell self-renewal have not yet been identified. The discovery of such factors is an important goal in gene therapy and spinal cord transplantation applications.

Briefly, polystyrene tissue culture untreated 96-well plates are coated with fn fragments at a coating concentration of 0.2 μg / cm ² . Mouse bone marrow cells are plated in 0.2 ml serum free medium (1,000 cells / well). Cells cultured in the presence of IL-3 (5 ng / ml) + SCF (50 ng / ml) act as a positive control under conditions where stem cell self-renewal is not expected and clear differentiation is expected.
The albumin fusion protein of the present invention is tested with an appropriate negative control in the presence or absence of SCF (5.0 ng / ml), wherein an administered composition comprising the albumin fusion protein of the present invention is tested. The volume represents 10% of the total assay volume. The plated cells are then grown by incubating for 7 days in a tissue culture incubator in a hypoxic environment (5% CO ₂ , 7% O ₂ and 88% N ₂ ). The number of proliferating cells in the well is then quantified by measuring thymidine incorporation into the cellular DNA. Confirmation of positive hits in the assay requires phenotypic characterization of the cell, which is achieved by scaling up the culture system and using appropriate antibody reagents against cell surface antigens and FACScan. .

  If a particular fusion protein of the invention is found to be a stimulant of hematopoietic ancestry, the fusion protein and polynucleotide corresponding to this fusion protein are useful in the diagnosis and treatment of disorders affecting the immune system and hematopoiesis. possible. Exemplary uses are described in the “Immune Activity” and “Infectious Diseases” sections above, and elsewhere herein. Fusion proteins may also be useful in the expansion of various blood chain stem cells and committed cells and in the differentiation and / or proliferation of various cell types.

  Furthermore, the albumin fusion proteins of the invention and polynucleotides encoding the fusion proteins of the invention are also used to inhibit hematopoietic cell proliferation and differentiation, and thereby, during chemotherapy, from chemotherapeutic agents Can be used to protect bone marrow stem cells. This anti-proliferative effect may allow for the administration of higher doses of chemotherapeutic agents and thus more effective chemotherapeutic treatment.

  Furthermore, the fusion proteins of the invention and polynucleotides encoding the albumin fusion proteins of the invention also treat and diagnose hematopoietic related disorders (eg, anemia, pancytopenia, leukopenia, thrombocytopenia, or leukemia). Can be useful to. This is because stromal cells are important in the generation of hematopoietic lineage cells. These uses include bone marrow cell ex vivo culture, bone marrow transplantation, bone marrow remodeling, neoplastic radiation therapy or chemotherapy.

Example 86: Proliferation of human skin fibroblasts and aortic smooth muscle cells
The fusion protein of the invention is added to a culture of normal human skin fibroblasts (NHDF) and human aortic smooth muscle cells (AoSMC), and two simultaneous assays are performed with each sample. The first assay tests the effect of the fusion protein on the proliferation of normal human fibroblasts (NHDF) or aortic smooth muscle cells (AoSMC). Abnormal proliferation of fibroblasts or smooth muscle cells is part of several pathological processes, including fibrosis and restenosis. The second assay tests IL6 production by both NHDF and SMC. IL6 production is an indicator of functional activation, activated cells have increased production of numerous cytokines and other factors, which results in proinflammatory or immunoregulatory results obtain. The assay is performed with and without co-TNFα stimulation to check for costimulatory or inhibitory activity.

  Briefly, on day 1, place 96 cells / well (NHDF) or 2000 cells / well (AoSMC) in 100 μl culture medium in a 96-well black plate. NHDF culture medium contains the following: Clonetics FB basal medium, 1 mg / ml hFGF, 5 mg / ml insulin, 50 mg / ml gentamicin, 2% FBS, while AoSMC culture medium includes: Clonetics SM basal medium, 0.5 μg / ml Contains ml hFGF, 5 mg / ml insulin, 1 μg / ml hFGF, 50 mg / ml gentamicin, 50 μg / ml Amphotericin B, 5% FBS. After incubation at 37 ° C. for at least 4-5 hours, the culture medium is aspirated and replaced with growth arrest medium. Growth stop medium for NHDF contains fibroblast basal medium, 50 mg / ml gentamicin, 2% FBS, while growth stop medium for AoSMC is SM basal medium, 50 mg / ml gentamicin, 50 μg / ml Amphotericin B, 0 Includes 4% FBS. Incubate at 37 ° C for up to 2 days.

On day 2, serial dilutions and templates of the albumin fusion proteins of the invention are designed so that they always include a media control and a known protein control. For both stimulation and inhibition experiments, the protein is diluted in growth arrest medium. For inhibition experiments, TNFa is added to a final concentration of 2 ng / ml (NHDF) or 5 ng / ml (AoSMC). Add 1/3 volume of medium containing the control or albumin fusion protein of the invention and incubate at 37 ° C./5% CO ₂ until day 5.

Transfer 60 μl from each well to another labeled 96 well plate, cover with plate sealer and store at 4 ° C. (for IL6 ELISA) until day 6. Aseptically add Alamar Blue in an amount equal to 10% of the culture volume (10 μl) to the remaining 100 μl in the cell culture plate. Return plate to incubator for 3-4 hours.
The fluorescence with excitation at 530 nm and emission at 590 nm is then measured using a CytoFluor. This gives growth stimulation / inhibition data.

  On day 5, IL-6 ELISA is performed by coating 96 well plates with 50-100 μl / well anti-human IL-6 monoclonal antibody diluted in PBS (pH 7.4) and incubating ON at room temperature .

  On day 6, empty the contents of the plate into the sink and blot on a paper towel. Prepare an assay buffer containing PBS containing 4% BSA. Plates are blocked with 200 μ / well Pierce Super Block blocking buffer in PBS for 1-2 hours, then the plates are washed with wash buffer (PBS, 0.05% Tween-20). Blot the plate on a paper towel. Subsequently, 50 μl / well of anti-human IL-6 monoclonal biotin-labeled antibody diluted to 0.50 mg / ml is added. Make dilutions in medium of IL-6 stock (30 ng / ml, 10 ng / ml, 3 ng / ml, 1 ng / ml, 0.3 ng / ml, 0 ng / ml). Duplicate samples are added to the top row of the plate. Cover the plate and incubate for 2 hours at room temperature on a shaker.

  Wash plate with wash buffer and blot on paper towel. EU labeled streptavidin is diluted 1: 1000 in assay buffer and 100 μl / well is added. Cover the plate and incubate at room temperature for 1 hour. The plate is again washed with wash buffer and blotted on a paper towel.

  Add 100 μl / well enhancement solution. Shake for 5 minutes. Read the plate with a Wallac DELFIA fluorometer. The readings from triplicate samples in each assay are tabulated and averaged.

  A positive result in this assay suggests AoSMC cell proliferation and suggests that the albumin fusion protein may be involved in dermal fibroblast proliferation and / or smooth muscle cell proliferation. A positive result also suggests many potential uses of the fusion protein and polynucleotides encoding the fusion protein. For example, inflammation and immune responses, wound healing, and angiogenesis, as described throughout this specification. In particular, the fusion protein can be used to promote wound healing and skin regeneration and angiogenesis (both blood and lymphatic vessels). Vascular growth can be used, for example, in the treatment of cardiovascular disease. In addition, fusion proteins that exhibit antagonistic activity in this assay may act as anti-angiogenesis (eg, anti-angiogenesis) to treat diseases, disorders, and / or conditions, including angiogenesis. Can be useful to. These diseases, disorders, and / or conditions, such as the following: malignant tumors, solid tumors, benign tumors (eg, hemangiomas, acoustic neuromas, neurofibromas, trachomas, and purulent granulomas); arthrosclerotic plaques plaque); ocular angiogenic diseases (eg, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal transplant rejection, neovascular glaucoma, post-lens fibroplasia, skin flush, retinoblastoma, uvea) Inflammation and pterygium of eyes (abnormal blood vessel growth)); rheumatoid arthritis; psoriasis; delayed wound healing; endometriosis; angiogenesis; granulation; hyperplastic scar (keloid); Nonfracture; scleroderma; trachoma; vascular adhesion; myocardial neovascularization; coronary collateral branch; cerebral collateral branch; arteriovenous malformation; Osler-Webber syndrome; plaque neovascularization; telangiectasia; hemophilia joint; angiofibroma; fibromyogenic dysplasia; wound granulation; Crohn's disease; and atherosclerosis Are known in the art and / or described herein. In addition, albumin fusion proteins that act as antagonists in this assay are useful in treating anti-hyperproliferative and / or anti-inflammatory diseases as known in the art and / or described herein. It can be.

(Example 87: Expression of cell adhesion molecule (CAM) on endothelial cells)
The recruitment of lymphocytes to areas of inflammation and angiogenesis involves specific receptor-ligand interactions between cell surface adhesion molecules (CAM) on lymphocytes and the vascular endothelium. The adhesion process follows a multi-stage cascade in both normal and pathological environments, which cascade is intracellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule on endothelial cells (EC) 1 (VCAM-1), and expression of endothelial leukocyte adhesion molecule 1 (E-selectin). Expression of these molecules, etc. on the vascular endothelium determines the efficiency with which leukocytes can adhere to the local vasculature and overflow into the local tissue during the progression of the inflammatory response. Local concentrations of cytokines and growth factors are involved in regulating the expression of these CAMs.

Briefly, endothelial cells (eg, human umbilical vein endothelial cells (HUVEC)) are grown to confluence in standard 96 well plates. Growth medium is removed from the cells and replaced with 100 μl of 199 Medium (10% fetal bovine serum (FBS)). Samples for testing (containing the albumin fusion protein of the invention) and positive or negative controls are added to triplicate (10 μl volumes) plates. The plates are then incubated at 37 ° C. for either 5 hours (selectin and integrin expression) or 24 hours (integrin expression only). Plates are aspirated to remove media and 100 μl 0.1% paraformaldehyde-PBS (containing Ca ++ and Mg ++) is added to each well. Hold plate at 4 ° C. for 30 minutes. The fixative is removed from the wells and the wells are washed once with PBS (+ Ca, Mg) + 0.5% BSA and drained. 10 μl of diluted primary antibody is added to the test and control wells. Anti-ICAM-1-biotin, anti-VCAM-1-biotin, and anti-E-selectin-biotin are used at a concentration of 10 μg / ml (1:10 dilution of 0.1 mg / ml stock antibody). Cells are incubated at 37 ° C. for 30 minutes in a humidified environment. The wells are washed 3 times with PBS (+ Ca, Mg) + 0.5% BSA. 20 μl of diluted ExtrAvidin-Alkaline Phosphate (1: 5,000 dilution, referred to herein as working diluent) is added to each well and incubated at 37 ° C. for 30 minutes. The wells are washed 3 times with PBS (+ Ca, Mg) + 0.5% BSA and drained. Dissolve 1 tablet of p-nitrophenol phosphate (pNPP) per 5 ml of glycine buffer (pH 10.4). 100 μl pNPP substrate in glycine buffer is added to each test well. Triplicate standard wells are prepared from working dilutions of ExtrAvidin-Alkaline Phosphatase in glycine buffer; 1: 5,000 (10 ⁰ )> 10 ^−0.5 > 10 ⁻¹ > 10 ^−1.5. . When 5 μl of each dilution is added to triplicate wells, the resulting AP content in each well is 5.5 ng, 1.74 ng, 0.55 ng, 0.18 ng. 100 μl of pNNP reagent is then added to each of the standard wells. The plate is incubated at 37 ° C. for 4 hours. A volume of 50 μl of 3M NaOH is added to all wells. The plate is read on a 405 nm plate reader using the background removal option in blank wells filled with glycine buffer only. In addition, the template is adjusted to indicate the concentration of AP conjugate in each standard well [5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results are shown as the amount of bound AP copolymer in each sample.

Example 88: Alamar Blue Endothelial Cell Proliferation Assay
This assay can be used to quantitatively determine protein-mediated inhibition of bFGF-induced proliferation of bovine lymphatic endothelial cells (LEC), bovine aortic endothelial cells (BAEC), or human microvascular myometrium (UTMEC). This assay incorporates a fluorometric growth indicator based on detection of metabolic activity. A standard Alamar Blue proliferation assay is prepared in EGM-2MV with 10 ng / ml bFGF added as a source of endothelial cell stimulation. This assay can be used with growth media and various endothelial cells with slight changes in cell concentration. Dilute the dilutions of the protein batch to be tested appropriately. Serum-free medium without bFGF (BIBCO SFM) is used as an unstimulated control and Angiostatin or TSP-1 is included as a known inhibition control.

  Briefly, LEC, BAEC or UTMEC are seeded in 96-well plates at a density of 5000-2000 cells / well in growth medium and left at 37 ° C. overnight. After overnight incubation of the cells, the growth medium is removed and replaced with GIBCO EC-SFM. The cells are treated in triplicate wells containing additional bFGF with an appropriate dilution of the albumin fusion protein of the invention or control protein sample (prepared in SFM) to a concentration of 10 ng / ml. Once the cells are treated with the sample, the plate (s) are returned to the 37 ° C. incubator for 3 days. After 3 days, 10 ml of stock alamar blue (Biosource Cat # DAL1100) is added to each well and the plate (s) is returned to the 37 ° C. incubator for 4 hours. The plate or plates are then read using a CytoFluor fluorescence reader with 530 nm excitation and 590 nm emission. Direct output is recorded in relative fluorescence units.

Alamar blue is an oxidation-reduction indicator that fluoresces and changes color in response to chemical reduction of the growth medium resulting from cell growth. As cells proliferate in culture, the natural metabolic activity causes a chemical reduction of the surrounding environment immediately. By reduction on growth, the indicator can change from an oxidized (non-fluorescent blue) form to a reduced (fluorescent red) form.
That is, stimulated growth produces a stronger signal, and inhibited growth produces a weaker signal, and the total signal is proportional to the total number of cells as well as their metabolic activity. A background level of activity is observed using only starvation medium. This is compared to the output observed from the positive control sample (bFGF in growth medium) and protein dilutions.

(Example 89: Detection of inhibition of mixed lymphocyte reaction)
This assay can be used to detect and assess inhibition of mixed lymphocyte reaction (MLR) by the fusion proteins of the invention. Inhibition of MLR may result from direct effects on cell proliferation and viability, regulation of costimulatory molecules in interacting cells, regulation of adhesion between lymphocytes and accessory cells, or regulation of cytokine production by accessory cells . Multiple cells can be targeted by an albumin fusion protein of the invention that inhibits MLR. This is because the peripheral blood mononuclear fraction used in this assay contains T lymphocytes, B lymphocytes and natural killer lymphocytes, as well as monocytes and dendritic cells.

  The albumin fusion proteins of the invention found to inhibit MLR may find use in diseases related to lymphocyte and monocyte activation or proliferation. This includes asthma, arthritis, diabetes, inflammatory skin conditions, psoriasis, eczema, systemic lupus erythematosus, multiple sclerosis, glomerulonephritis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, arteriosclerosis, Diseases such as, but not limited to, cirrhosis, graft-versus-host disease, graft-versus-host disease, hepatitis, leukemia and lymphoma.

Briefly, PBMCs derived from human donors are purified by density gradient centrifugation using Lymphocyte Separation Medium (LSM®, density 1.0770 g / ml, Organon Teknika Corporation, West Chester, PA). PBMCs from two donors are adjusted to 2 × 10 ⁶ cells / ml in RPMI-1640 (Life Technologies, Grand Island, NY) supplemented with 10% FCS and 2 mM glutamine. The PBMC from the third donor is adjusted to 2 × 10 ⁵ cells / ml. 50 μl of PBMC from each donor is added to the wells of a 96 well round bottom microtiter plate. A dilution (50 μl) of the fusion protein test material is added to the microtiter wells in triplicate. Add to test sample (of protein of interest) to a final dilution of 1: 4; add rhuIL-2 (R & D Systems, Minneapolis, Minn., Cat. No. 202-IL) to a final concentration of 1 μg / ml Anti-CD4 mAb (R & D Systems, clone 34930.11, catalog number MAB379) is added to a final concentration of 10 μg / ml. Cells are cultured for 7-8 days at 37 ° C. in 5% CO ₂ and 1 μC [ ³ H] thymidine is added to the wells for the last 16 hours of culture. Cells are harvested and thymidine incorporation is determined using a Packard TopCount. Data are expressed as the mean and standard deviation of triplicate measurements.

  Samples of the fusion protein of interest are screened in separate experiments and negative control treatment, anti-CD4 mAB (which inhibits lymphocyte proliferation), and positive control treatment, IL-2 (recombinant or above) As compared to (which promotes lymphocyte proliferation).

Example 90: Assay for protease activity
The following assay can be used to assess the protease activity of an albumin fusion protein of the invention.

  Gelatin and casein enzyme electrophoresis is performed essentially as described (Heusen et al., Anal. Biochem., 102: 196-202 (1980); Wilson et al., Journal of Urology, 149: 653-658 (1993). )). Samples were run on a 10% polyacrylamide / 0.1% SDS gel containing 1% gelatin or casein, soaked in 2.5% Triton for 1 hour at room temperature, and in 0.1 M glycine (pH 8.3) Soak at 37 ° C. for 5-16 hours. After staining in amide, the black areas of proteolysis appear as distinct areas against the dark blue background. Trypsin (Sigma T8642) is used as a positive control.

Protease activity is also determined by monitoring the cleavage of n-α-benzoyl-L-arginine ethyl ester (BAEE) (Sigma B-4500). The reaction is adjusted with (25 mM NaPO ₄ , 1 mM EDTA, and 1 mM BAEE), pH 7.5. Samples are added and the change in absorbance at 260nm is monitored in a time-drive mode using a Beckman DU-6 spectrophotometer. Trypsin is used as a positive control.

  A further assay is based on the release of acid soluble peptides from casein or hemoglobin measured either as absorbance at 280 nm or colorimetrically using the Folin method, Bergmeyer et al., Methods of Enzymatic Analysis, 5 (1984). As described in. Other assays involve solubilization of chromogenic substrates (Ward, Applied Science, 251-317 (1983)).

(Example 91: Identification of serine protease substrate specificity)
Methods known in the art or described herein can be used to determine the substrate specificity of an albumin fusion protein of the invention having serine protease activity. A preferred method for determining substrate specificity is by the use of positional scanning of synthetic combinatorial libraries, as described in GB 2 324 529, which is incorporated herein in its entirety.

Example 92 Ligand Binding Assay
The following assay can be used to assess the ligand binding activity of the albumin fusion proteins of the invention.

  Ligand binding assays provide a direct method for confirming receptor pharmacology and are adaptable to a fast format. The purified ligand for the albumin fusion protein of the invention is radioisotopically labeled to a high specific activity (50-2000 Ci / mmol) for binding studies. A determination is then made so that the process of radioisotope labeling does not reduce the activity of the ligand for the fusion protein. Assay conditions for buffers, ions, pH and other modulators (eg, nucleotides) are optimized to construct effective signal-to-noise ratios for both membrane and whole-cell polypeptide sources. For these assays, specific polypeptide binding is defined as the difference between the total associated radioactivity minus the radioactivity measured in the presence of excess unlabeled competing ligand. Where possible, more than one competing ligand is used to define residual non-specific binding.

Example 93: Functional assay in Xenopus oocytes
A cap RNA transcript derived from a linearized plasmid template encoding an albumin fusion protein of the invention is synthesized in vitro using RNA polymerase according to standard procedures. In vitro transcripts are suspended in water at a final concentration of 0.2 mg / ml. The ovarian lobe is removed from the adult female frog, stage V follicle-depleted oocytes are obtained, and the RNA transcript (10 ng / oocyte) is injected in a 50 nl bolus using a microinjector. A two-electrode voltage clamp is used to measure currents from individual Xenopus oocytes to fusion protein and polypeptide agonist exposure. Recordings are made in Barth's medium without Ca 2+ at room temperature. This Xenopus system can be used for screening of known ligands and tissue / cell extracts for ligand activation.

Example 94 Microphysiomeric Assay
Activation of a wide variety of secondary messenger systems results in the ejection of small amounts of acid from the cells. The acid formed is primarily the result of the increased metabolic activity necessary to stimulate intracellular signaling processes. Although the pH change of the medium surrounding the cells is very small, it can be detected by a CYTOSENSOR microphysiology measuring device (Molecular Devices Ltd., Menlo Park, Calif.). Thus, CYTOSENSOR can detect the ability of the albumin fusion proteins of the present invention to activate second messengers that couple to energy-mediated intracellular signaling systems.

(Example 95: Extract / cell supernatant screening)
There are a large number of mammalian receptors for which no cognate activating ligands (agonists) exist. Thus, active ligands for these receptors may not be included in the ligand bank as identified to date. Thus, the albumin fusion protein of the present invention can also be used against tissue extracts (calcium, cAMP) to identify the therapeutic protein portion of the albumin fusion protein and / or the natural ligand for the albumin protein portion. Functional screening using functional screening such as microphysiology instruments, oocyte electrophysiology, etc. Extracts that produce a positive functional response can be successively subdivided until the activating ligand is isolated and identified.

Example 96: ATP binding assay
The following assays can be used to evaluate ATP binding assays of the fusion proteins of the invention.

The ATP binding activity of the albumin fusion proteins of the invention can be detected using the ATP binding assay described in US Pat. No. 5,858,719, which is incorporated herein by reference in its entirety. Briefly, ATP binding to the albumin fusion protein of the invention is measured via photoaffinity labeling with 8-azido-ATP in a competition assay. The reaction mixture containing 1 mg / ml ABC transport protein is incubated with various concentrations of ATP or non-hydrolyzable ATP analog adenyl-5′-imidonilic acid at 4 ° C. for 10 minutes. A mixture of 8-azido-ATP (Sigma Chem. Corp., St. Louis, MO.) Plus 8-azido-ATP ( ³² P-ATP) (5 mCi / Lmol, ICN, Irvine CA.) was added to 100 μM. Add to final concentration and place 0.5 ml aliquot into porcelain spotplate wells on ice. The plate is irradiated twice at a distance of 2.5 cm from the plate using a short wave 254 nm UV lamp at 1 minute intervals (with a 1 minute cooling interval in between). The reaction is stopped by adding dithiothreitol to a final concentration of 2 mM. This incubation is subjected to SDS-PAGE electrophoresis, drying, and autoradiography. The protein band corresponding to the albumin fusion protein of the present invention is cut out and the radioactivity is quantified. A decrease in radioactivity by increasing ATP or adenyl-5'-imidiniphosphate provides a measure of ATP affinity for the fusion protein.

Example 97: Phosphorylation assay
For assays for phosphorylation activity of the albumin fusion proteins of the present invention, phosphorylation as described in US Pat. No. 5,958,405, which is incorporated herein by reference. Utilize the assay. Briefly, phosphorylation activity can be measured by phosphorylating protein substrates using γ-labeled ³² P-ATP and quantifying the incorporated radioactivity using a γ radioisotope counter. . The fusion protein of the invention is incubated with protein substrate, ³² P-ATP and kinase buffer. The ³² P incorporated into the substrate is then separated from free ³² P-ATP by electrophoresis, the incorporated ³² P is counted and compared to a negative control. A radioactivity count greater than the negative control indicates phosphorylation activity of the fusion protein.

(Example 98: Detection of phosphorylation activity (activation) of albumin fusion protein of the present invention in the presence of polypeptide ligand)
Methods known in the art or described herein can be used to determine the phosphorylation activity of the albumin fusion proteins of the present invention. A preferred method of determining phosphorylation activity uses a tyrosine phosphorylation assay, as described in US Pat. No. 5,817,471 (incorporated herein by reference).
(Example 99: Identification of a signal transduction protein that interacts with the albumin fusion protein of the present invention)
The albumin fusion proteins of the invention can serve as a research tool for the identification, characterization and purification of signal transduction pathway proteins or receptor proteins. Briefly, the labeled fusion protein of the invention is useful as a reagent for purification of the molecule with which it interacts. In one embodiment of affinity purification, the albumin fusion protein of the invention is covalently bound to a chromatography column. A cell-free extract derived from a putative target cell (eg, cancer tissue) is passed through the column to bind a molecule with appropriate affinity to the albumin fusion protein. The protein complex is recovered from the column, separated, and the recovered molecule is subjected to N-terminal protein sequencing. This amino acid sequence is then used to identify complementary molecules or to design degenerate oligonucleotide probes for cloning related genes from appropriate cDNA libraries.

Example 100: IL-6 bioassay
Various assays for testing the proliferative effects of the albumin fusion proteins of the present invention are known in the art. For example, one such assay is described by Marz et al. (Proc. Natl. Acad Sci., USA, 95: 3251-56 (1998), incorporated herein by reference). As is the IL-6 bioassay. After 68 hours at 37 ° C., the number of viable cells is determined by adding the tetrazolium salt thiazolyl blue (MTT) and further incubating at 37 ° C. for 4 hours. B9 cells are lysed by SDS and the optical density is measured at 570 nm. Control with IL-6 (positive) and control without cytokine (negative). Briefly, IL-6 dependent mouse B9 cells were washed 3 times in medium without IL-6, plated at a concentration of 5,000 cells / well in 50 μl, and 50 μl of the fusion of the invention. Add protein. Use. Proliferation in a test sample (including an albumin fusion protein of the present invention) enhanced compared to a negative control indicates a proliferative effect mediated by the fusion protein.

Example 101: Supporting the survival of chicken embryo neurons
To test whether sympathetic cell viability is supported by the albumin fusion protein of the present invention, Senaldi et al.'S chicken embryo neuron survival assay can be utilized (Proc. Natl. Acad. Sci., U.S.). S.A., 96: 11458-63 (1998), which is incorporated herein by reference). Briefly, motor neurons and sympathetic nerves are isolated from chicken embryos and L15 medium (containing 10% FCS, glucose, sodium selenite, progesterone, conalbumin, putrescine, and insulin; Life Technologies, Rockville, MD.) And Dulbecco's modified Eagle's medium [including 10% FCS, glutamine, penicillin, and 25 mM Hepes buffer (pH 7.2); Life Technologies, Rockville, MD. ], Each resuspended and in the presence of different concentrations of the purified fusion protein of the invention, as well as the negative control without any cytokines and incubated at 37 ° C. in 5% CO ₂ . After 3 days, neuronal survival is determined by evaluation of cell morphology using the Mosmann colorimetric assay (Mosmann, T., J. Immunol. Methods, 65: 55-63 (1983)). Enhanced neuronal cell viability compared to cytokine-free controls indicates the ability of the albumin fusion protein to increase neuronal cell survival.

Example 102: Assay for phosphatase activity
The following assay can be used to assess the serine / threonine phosphatase (PTPase) activity of albumin fusion proteins of the invention.

Assays widely known to those skilled in the art can be utilized to assay for serine / threonine phosphatase (PTPase) activity. For example, the serine / threonine phosphatase (PSPase) activity of the albumin fusion proteins of the present invention is described in New England Biolabs, Inc. It can be measured using a manufactured PSPase assay kit. Myelin basic protein (MyBP) (a substrate for PSPase) is phosphorylated at serine and threonine residues using cAMP-dependent protein kinase in the presence of [ ³² P] ATP. Protein serine / threonine phosphatase activity is then determined by measuring the release of inorganic phosphate from 32P-labeled MyBP.

  Example 103: Serine / threonine phosphatase interaction with other proteins Fusion proteins of the invention having serine / threonine phosphatase activity (eg, as determined in Example 102) can be further interacted, for example. It is useful as a research tool for the identification, characterization and purification of proteins or receptor proteins or other signaling pathway proteins. Briefly, the labeled fusion protein of the present invention is useful as a reagent for the purification of molecules that interact with the fusion protein. In one embodiment of affinity purification, the albumin fusion protein of the invention is covalently bound to a chromatography column. A cell-free extract (eg, neuronal or hepatocyte) from the putative target cell passes through the column and molecules with the appropriate affinity bind to the fusion protein. The fusion protein-complex is recovered from the column, separated, and the recovered molecule is subjected to N-terminal protein sequencing. This amino acid sequence is then used to identify a capture molecule or to design a degenerate oligonucleotide probe for cloning related genes from an appropriate cDNA library.

Example 104: Assay for heparanase activity
There are a number of assays known in the art that can be used in assays for heparanase activity of the albumin fusion proteins of the present invention. In one example, the heparanase activity of an albumin fusion protein of the invention is assayed as described in Vlodavsky et al. (Vlodavsky et al., Nat. Med., 5: 793-802 (1999)). Briefly, cell lysates, conditioned media, intact cells (1 × 10 ⁶ cells / 35 mm dish), cell culture supernatant, or purified fusion protein are incubated at 37 ° C. for 18 hours at pH 6.2-6.6. Incubate with ³⁵ S-labeled ECM or soluble ECM-induced peak I proteoglycans. The incubation medium is centrifuged and the supernatant is analyzed by gel filtration using a Sepharose CL-6B column (0.9 × 30 cm). Fractions are eluted with PBS and their radioactivity is measured. The heparan sulfate side chain degradation fragment is eluted from Sepharose 6B with 0.5 <K _av <0.8 (Peak II). Each experiment is performed at least three times. The fragment corresponding to “Peak II” shows the activity of the albumin fusion protein of the present invention in the cleavage of heparan sulfate, as described by Vlodavsky.

(Example 105: Immobilization of biomolecule)
This example describes a method for the stabilization of an albumin fusion protein of the invention in a non-host cell lipid bilayer construct (eg, Bieri et al., Nature Biotech 17: 1105-1108 (1999)) Which is incorporated herein by reference), and this method can be adapted for the study of the fusion proteins of the invention in the various functional assays described above. Briefly, carbohydrate specific chemistry for biotinylation is used to bind a biotin tag to the albumin fusion protein of the present invention, allowing a uniform orientation during immobilization. A 50 μM solution of an albumin fusion protein of the invention in a washed membrane, together with 20 mM NaIO ⁴ , 1.5 mg / ml (4 mM) BACH or 2 mg / ml (7.5 mM) biotin-hydrazide (reaction volume, 150 μl), Incubate for 1 hour at room temperature. The sample was then subjected to dialysis (Pierce Sliderizer Cassett, 10 kDa cutoff; Pierce Chemical Co., Rockford IL) with a buffer exchange every 5 hours at 4 ° C. for 1 hour, and finally a 500 ml buffer. Dialyze with Solution R (0.15 M NaCl, 1 mM MgCl ₂ , 10 mM sodium phosphate, pH 7) for 12 hours. Immediately before addition to the cuvette, the sample is diluted 1: 5 with buffer ROG50 (buffer R supplemented with 50 mM octylglucoside).

Example 106: Assay for metalloproteinase activity
Metalloproteinases are peptide hydrolases that use metal ions (eg, Zn ²⁺ ) as a catalytic mechanism. The metalloproteinase activity of the albumin fusion protein of the invention can be assayed according to methods known in the art. The following exemplary methods are provided:
(Proteolysis of α-2-macroglobulin)
In order to confirm proteinase activity, the purified fusion protein of the invention was added to 1 × assay buffer (50 mM HEPES, pH 7.5, 0.2 M NaCI, 10 mM CaCl ₂ , 25 μM ZnCl ₂ and 0.05% Brij-35). Mix with substrate α-2-macroglobulin (0.2 units / ml; Boehringer Mannheim, Germany) and incubate at 37 ° C. for 1-5 days. Trypsin is used as a positive control. The negative control contains only α-2-macroglobulin in the assay buffer. Samples are collected, boiled in SDS-PAGE sample buffer containing 5% 2-mercaptoethanol for 5 minutes and then loaded onto an 8% SDS-polyacrylamide gel. After electrophoresis, these proteins are visualized by silver staining. Proteolysis is evident by the appearance of a low molecular weight band compared to the negative control.

(Inhibition of α-2-macroglobulin proteolysis by metalloproteinase inhibitors) Known metalloproteinase inhibitors (metal chelating agents (EDTA, EGTA and HgCl ₂ ), peptide metalloproteinase inhibitors (TIMP-1 and TIMP-2)) , And commercially available small molecule MMP inhibitors) can also be used to characterize the albumin fusion protein proteolytic activity of the present invention. Three synthetic MMP inhibitors that can be used are: MMP inhibitor I, “IC ₅₀ = 1.0 μM for MMP-1 and for MMP-8; IC ₅₀ = 30 μM for MMP-9; MMP− IC ₅₀ = 150 μM for 3; MMP-3 (Stromelysin-1) inhibitor I “IC ₅₀ = 5 μM for MMP-3” and MMP-3 inhibitor II “K _i for MMP-3” = 130 nM "; inhibitors available through Calbiochem (catalog numbers 444250, 444218 and 444225, respectively). Briefly, different concentrations of small molecule MMP inhibitors were combined with purified fusion protein of the invention (50 μg / ml) and 22.9 μl 1 × HEPES buffer (50 mM HEPES, pH 7.5, 0.2 M NaCl, 10 mM CaCl ₂ , 25 μM). ZnCl ₂ and 0.05% Brij-35), incubated for 2 hours at room temperature (24 ° C.), then 7.1 μL of substrate α-2-macroglobulin (0.2 units / ml) Add and incubate at 37 ° C. for 20 hours. The reaction is stopped by the addition of 4X sample buffer and immediately boiled for 5 minutes. After SDS-PAGE, the protein band is visualized by silver staining.

(Synthetic fluorogenic peptide substrate cleavage assay)
Substrate specificity for fusion proteins of the invention having exemplified metalloproteinase activity can be determined using techniques known in the art (eg, synthetic fluorogenic peptide substrates (purchased from BACHEM Bioscience Inc)). Test substrates include M-1985, M-2225, M-2105, M-2110 and M-2255. The first four substrates are MMP substrates, and the last one is a tumor necrosis factor-α (TNF-α) convertase (TACE) substrate. These substrates are preferably prepared in 1: 1 dimethyl sulfoxide (DMSO) and water. The stock solution is 50-500 μM. The fluorescence assay is performed using a Perkin Elmer LS 50B luminescence spectrometer equipped with a constant temperature water bath. The excitation λ is 328 nm and the emission λ is 393 nm. Briefly, this assay was performed using 176 μl 1 × HEPES buffer (0.2 M NaCl, 10 mM CaCl ₂ , 0.05% Brij-35 and 50 mM HEPES, pH 7.5) with 4 μl substrate solution (50 μM) at 25 ° C. This is done by incubating for 15 minutes, and then 20 μl of the purified fusion protein of the invention is added to the assay cuvette. The final concentration of substrate is 1 μM. The initial hydrolysis rate is monitored for 30 minutes.

Example 107: Identification and cloning of VH and VL domains
One method for identifying and cloning VH and VL domains from a cell line expressing a particular antibody is to use VH-specific primers and VL-specific primers for cDNA generated from the cell line expressing the antibody. PCR is performed using primers. Briefly, RNA is isolated from cell lines and used as a template for RT-PCR designed to amplify the VH and VL domains of antibodies expressed by EBV cell lines. Cells can be lysed with TRIzol® reagent (Life Technologies, Rockville. MD) and extracted with 1/5 volume of chloroform. Following the addition of chloroform, the solution is incubated at room temperature for 10 minutes and then centrifuged at 14,000 rpm for 15 minutes at 4 ° C. in a tabletop centrifuge. The supernatant is collected and the RNA is precipitated with an equal volume of isopropanol. Precipitated RNA is pelleted by centrifuging at 14,000 rpm for 15 minutes at 4 ° C. in a tabletop centrifuge. After centrifugation, the supernatant is discarded and washed with 75% ethanol. After washing, the RNA is again centrifuged at 800 rpm for 5 minutes at 4 ° C. Discard the supernatant and air dry the pellet. RNA is dissolved in DEPC water and heated at 60 ° C. for 10 minutes. The amount of RNA can be determined using optical density measurements.

According to methods well known in the art, cDNA can be synthesized from 1.5 μg to 2.5 μg of RNA using reverse transcriptase and random hexamer primers. The cDNA is then used as a template for PCR amplification of VH and VL domains. The primers used to amplify the VH and VL genes are shown in Table 7. Typically, a PCR reaction uses a single 5 'primer and a single 3' primer. Sometimes 5 'primer groups and / or 3' primer groups may be used when the amount of RNA template available is limited or for higher efficiency. For example, sometimes all five VH-5 ′ primers and all JH3 ′ primers are used in a single PCR reaction.
The PCR reaction is performed in a 50 μl volume containing 1 × PCR buffer, 2 mM of each dNTP, 0.7 units of High Fidelity Taq polymerase, 5 ′ primer mix, 3 ′ primer mix and 7.5 μl cDNA. Both VH and VL 5'primer and 3'primer mixtures can be made by pooling together 22 pmole individual primers and 28 pmole individual primers, respectively. PCR conditions are: 96 ° C. for 5 minutes; then 25 cycles of 94 ° C. for 1 minute, 50 ° C. for 1 minute, and 72 ° C. for 1 minute; followed by an extension cycle at 72 ° C. for 10 minutes. After the reaction is complete, store the sample tube at 4 ° C.

次いで、ＰＣＲサンプルを１．３％のアガロースゲルで電気泳動する。予測するサイズのＤＮＡバンド（ＶＨドメインについて約約５０６塩基対、およびＶＬドメインについて３４４塩基対）を、ゲルから切り出し得、そして、当該分野で周知の方法を用いて精製し得る。精製ＰＣＲ産物を、ＰＣＲクローニングベクター（Ｉｎｖｉｔｒｏｇｅｎ Ｉｎｃ．、Ｃａｒｌｓｂａｄ、ＣＡからのＴＡベクター）へ連結し得る。Ｅ．ｃｏｌｉのトランスフェクションおよび青色／白色の選別後、別個のクローン化ＰＣＲ産物を単離し得る。次いで、クローン化されたＰＣＲ産物を、当該分野で一般に公知の方法を用いて配列決定し得る。

The PCR sample is then electrophoresed on a 1.3% agarose gel. A DNA band of the expected size (about 506 base pairs for the VH domain and 344 base pairs for the VL domain) can be excised from the gel and purified using methods well known in the art. The purified PCR product can be ligated into a PCR cloning vector (TA vector from Invitrogen Inc., Carlsbad, CA). E. After transfection of E. coli and blue / white sorting, a separate cloned PCR product can be isolated. The cloned PCR product can then be sequenced using methods generally known in the art.

  PCR bands containing VH and VL domains can also be used to create full length Ig expression vectors. The VH and VL domains can be cloned into a vector containing the nucleotide sequence of the heavy chain (eg, human IgG1 or human IgG4) constant region or light chain (human κ or human λ) constant region and into a suitable host cell. When transfected, full heavy chain molecules or full light chain molecules can be expressed from these vectors. Furthermore, if the cloned heavy and light chains are both expressed in one cell line (either from one vector or two vectors) they are fully functional and secreted into the cell culture medium. Can be assembled into antibody molecules. Methods for using polynucleotides encoding VH antibody domains and VL antibody domains to generate expression vectors encoding complete antibody molecules are well known in the art.

(Example 108: Construct ID 2672, HSA-T20, preparation)
Construct ID 2672 (SEQ ID NO: 1186), pSAC35: HSA. T20 is yeast S. cerevisiae. cerevisiae expression vector pSAC35 contains DNA encoding a T20 albumin fusion protein having a full length HSA fused to the amino terminus of the HIV-1 inhibitory peptide T20 (ie, Y643-F678). The T20 peptide is derived from the ectodomain of the HIV-1 transmembrane protein gp41, which has been shown to have inhibitory activity against HIV-1 infection.

(Cloning of T20 cDNA)
A polynucleotide encoding T20 was generated by PCR using the four overlapping primers T20-1, T20-2, T20-3 and T20-4 described below. This sequence is the yeast S. cerevisiae. Codon optimized for expression in C. cerevisiae. The PCR fragment was digested with Bsu 36I / Asc I and ligated into Bsu 36I / Asc I digested pScNHSA. The Not I fragment was then subcloned into the pSAC35 plasmid. Construct ID number 2672 encodes an albumin fusion protein comprising full length HSA and HIV-1 inhibitory peptide T20 (ie, Tyr-643-Phe-678 (SEQ ID NO: 1188)).

  4 overlapping oligonucleotide 5 ′ primers suitable for PCR amplification of polynucleotides encoding HIV-1 inhibitory peptide T20, HIV-1 inhibitory peptide T20-1 and HIV-1 inhibitory peptide T20-4 and A 3 ′ primer was synthesized:

Ｔ２０−１は、Ｂｓｕ ３６Ｉクローニング部位（下線で表記）、およびＨＳＡ成熟形態（配列番号１０３８）の最後の４つのアミノ酸残基をコードするヌクレオチド、およびＨＩＶ−１阻害ペプチドＴ２０の最初の８アミノ酸残基（すなわち、Ｔｙｒ−６４３からＬｅｕ−６５０）をコードする２４ヌクレオチド（太字で表記）へ取り込む。Ｔ２０−４において、Ａｓｃ Ｉ部位に下線を付しており、そして最後の３１ヌクレオチド（太字で表記）は、ＨＩＶ−１阻害ペプチドＴ２０の最後の１０アミノ酸残基（Ａｓｐ−６６９からＰｈｅ−６７８）をコードするＤＮＡの逆相補体である。Ｔ２０−２オリゴヌクレオチドおよびＴ２０−３オリゴヌクレオチドは、互いにオーバーラップし、そして各々、Ｔ２０−１およびＴ２０−４とオーバーラップしそしてＨＩＶ−１阻害ペプチドＴ２０をコードする。ＰＣＲ産物を精製し（例えば、Ｗｉｚａｒｄ ＰＣＲ Ｐｒｅｐｓ ＤＮＡ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｓｙｓｔｅｍ（Ｐｒｏｍｅｇａ Ｃｏｒｐ）を使用）、次いで、Ｂｓｕ３６ＩおよびＡｓｃＩで消化した。さらに、ゲル電気泳動によるＢｓｕ３６Ｉ−ＡｓｃＩフラグメントの精製後、この産物をＢｓｕ３６Ｉ／ＡｓｃＩで消化したｐＳｃＮＨＳＡへクローン化した。配列を確認した後、このＴ２０アルブミン融合タンパク質をコードする発現カセットを、Ｎｏｔ ＩフラグメントとしてｐＳＡＣ３５へサブクローンした。さらに、Ｎｏｔ Ｉフラグメントを、ｐＳＡＣ３５へサブクローンして構築物ＩＤ番号２６７２を得た。

T20-1 is the Bsu 36I cloning site (indicated underlined), the nucleotide encoding the last 4 amino acid residues of the HSA mature form (SEQ ID NO: 1038), and the first 8 amino acid residues of HIV-1 inhibitory peptide T20 Incorporate into the 24 nucleotides (shown in bold) encoding the group (ie, Tyr-643 to Leu-650). In T20-4, the Asc I site is underlined and the last 31 nucleotides (shown in bold) are the last 10 amino acid residues of the HIV-1 inhibitory peptide T20 (Asp-669 to Phe-678). Is the reverse complement of DNA encoding. T20-2 and T20-3 oligonucleotides overlap each other and overlap T20-1 and T20-4, respectively, and encode HIV-1 inhibitory peptide T20. The PCR product was purified (eg, using the Wizard PCR Preps DNA Purification System (Promega Corp)) and then digested with Bsu36I and AscI. Furthermore, after purification of the Bsu36I-AscI fragment by gel electrophoresis, this product was cloned into pScNHSA digested with Bsu36I / AscI. After confirming the sequence, the expression cassette encoding this T20 albumin fusion protein was subcloned into pSAC35 as a Not I fragment. In addition, the Not I fragment was subcloned into pSAC35, resulting in construct ID number 2672.

  Furthermore, analysis of the N-terminus of the albumin fusion protein expressed by amino acid sequencing can confirm the presence of the predicted HSA sequence (see below).

  The T20 albumin fusion protein of the present invention is preferably a mature form of HSA (ie, fused to either the N-terminus or C-terminus of HIV-1 inhibitory peptide T20 (ie, Tyr-643 to Phe-678) Asp-25 to Leu-609). In one embodiment of the invention, the T20 albumin fusion protein of the invention further contains a signal sequence that directs the nascent fusion protein in the secretory pathway of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed and the mature T20 albumin fusion protein is secreted directly into the culture medium. The T20 albumin fusion protein of the present invention may comprise a heterologous signal sequence, including MAF, INV, Ig, fibrin B, clusterin, insulin-like growth factor binding protein 4, a variant HSA leader sequence (including chimeric HSA / MAF leader sequences). But is not limited to) or other heterologous signal sequences known in the art. In a preferred embodiment, the T20 albumin fusion protein of the invention comprises the native HIV-1 transmembrane protein gp41 signal sequence. In a further preferred embodiment, the T20 albumin fusion protein of the present invention further comprises a methionine residue at the N-terminus. Polynucleotides encoding these polypeptides (including fragments and / or variants) are also encompassed by the present invention.

(Expression and purification of construct ID2672)
(Expression in yeast S. cerevisiae)
Construct 2672 is obtained from yeast S. cerevisiae by methods known in the art (see Example 3). can be transformed into cerevisiae. Expression levels can be tested by immunoblot detection using anti-HSA serum as the primary antibody.

(Purification from the supernatant of yeast S. cerevisiae cells)
Yeast S. In cerevisiae, the cell supernatant containing the secreted T20 albumin fusion protein expressed from construct ID number 2672 can be purified as described in Example 4. Sequencing of the N-terminus of the albumin fusion protein yields the sequence DAHKS (SEQ ID NO: 2143), corresponding to the amino terminus of the HSA mature form.

(T20 activity can be assayed using in vitro infectivity assays and / or cell-cell fusion inhibition assays)
In vitro infectivity assays and cell-cell fusion inhibition assays are described in Wild et al., “Peptides corresponding to a predictive alpha-helical domain of human immunofi cient in vitro pipe type 41 gp. Natl. Acad. Sci. USA, 91: 9770-9774 (1994).

(Method)
High titer virus stocks are described above (Wild, C., et al., “A synthetic peptide inhibitor of human immunogenicity 37 inc. USN. Proceedings of the United States. Proc. 1992)) and can be prepared in CEM human leukemia cells. Infectious titers can be assessed by end point dilution in AA5 and CEM continuous cell lines. Reverse transcriptase (RT) activity present in the supernatant can be received as a basis for successful infection. 50% tissue culture infectious dose (TCID ₅₀₎ is, Reed and Muench (Wild et al., "Peptides corresponding to a predictive alpha-helical domain of human immunodeficiency virus type 1 gp41 are potent inhibitors of virus infection ", Proc.Natl.Acad. Sci. USA, 91: 9770-9774 (1994)). Primary HIV-1 isolates can be grown in activated peripheral blood mononuclear cells (“PBMC”) from normal donors.

The ability of a T20 albumin fusion protein to inhibit infection of a basic cell-free virus (ie, HIV-1 _LAI or HIV-1 _NIHZ ), cell free containing various concentrations of T20 albumin fusion protein Can be evaluated by incubating serial dilutions of viruses with AA5 or CEM target cells. T20 albumin fusion protein can be tested in a similar assay using PBMC as target cells against the primary isolate and this basic HIV-1 _LAI isolate. Both assays are performed as described in Wild et al., 1992.

The ability of T20 albumin fusion protein to block virus-mediated cell-cell fusion can be assessed as previously described in Wild et al., 1992. Briefly, approximately 7 × 10 ⁴ MOLT-4 cells can be cultured with 10 ⁴ CEM cells and in 100 μL culture medium in a 96 well plate (plate where half of the region is a cluster; Costar). Can chronically infect HIV-1 isolates. 10 μL of T20 albumin fusion protein can be added and the cell mixture can be incubated for 24 hours at 37 ° C. At this time, multinucleated giant cells can be determined by microscopic examination at 40 magnifications.

(The activity of T20 albumin fusion encoded by Construct ID No. 2672 can be assessed using in vitro infectivity assays and / or cell-cell fusion inhibition assays)
(Method)
The T20 albumin fusion protein encoded by construct 2672 is as described above under the subsection title "T20 activity can be assayed using an in vitro infectivity assay and / or cell-cell fusion inhibition assay". Can be tested in in vitro infectivity bioassays and cell-cell fusion inhibition assays.

(Example 109: Generation of construct ID 2673, T20-HSA)
Construction ID 2673 (pSAC35: T20.HSA) is the yeast S. cerevisiae strain. T20 albumin with HSA chimeric leader sequence (ie HSA-kex2 signal peptide) followed by HIV-1 inhibitory peptide T20 (ie Y643-F678) fused to the amino terminus of the mature form of HSA in the cerevisiae expression vector pSAC35 Contains DNA encoding the fusion protein.

(Cloning of T20 cDNA)
DNA encoding the HIV-1 inhibitory peptide was PCR generated using four overlapping primers. This sequence is referred to as yeast S. cerevisiae. Codon optimized for expression in C. cerevisiae. The PCR fragment was digested with Sal I / Cla I and subcloned into pScCHSA digested with Xho I / Cla I. The Not I fragment was then subcloned into the pSAC35 plasmid. Construct ID number 2673 encodes the chimeric leader sequence of HSA fused to HIV-1 inhibitory peptide T20 (ie, Tyr-643-Phe-678) followed by the mature form of HSA.

  Four overlapping oligonucleotide 5 'and 3' primers (T20-5 and T20-6) suitable for PCR amplification of a polynucleotide encoding the HIV-1 inhibitory peptide T20 were synthesized.

Ｔ２０−５は、Ｓａｌ Ｉクローニング部位（下線で示される）、ＨＳＡキメラリーダー配列の最後の３アミノ酸残基をコードするヌクレオチド、およびＨＩＶ−１阻害性ペプチドＴ２０の最初の８アミノ酸（太字で示される）（すなわち、Ｔｙｒ−６４３〜Ｌｅｕ−６５０）をコードするＤＮＡを組み込む。Ｔ２０−６において、下線の配列はＣｌａ Ｉ部位であり；そしてこのＣｌａ Ｉ部位およびこれに続くＤＮＡは、成熟ＨＳＡタンパク質（配列番号１０３８）の最初の１０アミノ酸をコードするＤＮＡの逆相補体である。太字の配列は、ＨＩＶ−１阻害性ペプチドＴ２０の最後の１０アミノ酸残基（Ａｓｐ−６６９〜Ｐｈｅ−６７８）をコードする３１ヌクレオチドの逆相補体である。Ｔ２０−２およびＴ２０−３オリゴヌクレオチド（実施例１０８におけるような）は互いに、そしてそれぞれＴ２０−５およびＴ２０−６と重複し、そしてＨＩＶ−１阻害性ペプチドＴ２０をコードする。これらのプライマーを使用して、ＨＩＶ−１阻害性ペプチドＴ２０を、アニーリング、アニーリングされたプライマーの伸長、Ｓａｌ ＩおよびＣｌａ Ｉでの消化、ならびにＸｈｏ Ｉ／Ｃｌａ Ｉで消化されたｐＳｃＣＨＳＡへのサブクローニングによって生成した。配列を確認した後、Ｔ２０アルブミン融合発現カセットを含むＮｏｔ Ｉフラグメントを、構築物ＩＤ２６７３を生成するためにＮｏｔ Ｉで切断したｐＳＡＣ３５にサブクローニングした。構築物ＩＤ番号２６７３は、キメラリーダー配列、ＨＩＶ−１阻害性ペプチドＴ２０、およびＨＳＡの成熟形態を含むアルブミン融合タンパク質をコードする。

T20-5 is the Sal I cloning site (shown underlined), the nucleotide encoding the last 3 amino acid residues of the HSA chimeric leader sequence, and the first 8 amino acids of the HIV-1 inhibitory peptide T20 (shown in bold) ) (Ie, incorporating Tyr-643-Leu-650). In T20-6, the underlined sequence is a Cla I site; and this Cla I site and the DNA that follows is the reverse complement of DNA encoding the first 10 amino acids of the mature HSA protein (SEQ ID NO: 1038). . The bold sequence is a 31 nucleotide reverse complement that encodes the last 10 amino acid residues (Asp-669 to Phe-678) of the HIV-1 inhibitory peptide T20. T20-2 and T20-3 oligonucleotides (as in Example 108) overlap each other and T20-5 and T20-6, respectively, and encode the HIV-1 inhibitory peptide T20. Using these primers, HIV-1 inhibitory peptide T20 can be obtained by annealing, extension of the annealed primer, digestion with Sal I and Cla I, and subcloning into pScCHSA digested with Xho I / Cla I. Generated. After confirming the sequence, the Not I fragment containing the T20 albumin fusion expression cassette was subcloned into pSAC35 cut with Not I to generate construct ID2673. Construct ID number 2673 encodes an albumin fusion protein comprising a chimeric leader sequence, HIV-1 inhibitory peptide T20, and a mature form of HSA.

  Furthermore, analysis of the N-terminus of the expressed albumin fusion protein by amino acid sequencing can confirm the presence of the expected T20 sequence (see below).

  The T20 albumin fusion protein of the present invention is preferably a mature form of HSA (ie Asp) fused to either the N-terminus or C-terminus of HIV-1 inhibitory peptide T20 (ie Tyr-643-Phe-678). -25 to Leu-609). In one embodiment of the invention, the T20 albumin fusion protein of the invention further comprises a signal sequence that directs the nascent fusion polypeptide in the secretory pathway of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed and the mature T20 albumin fusion protein is secreted directly into the culture medium. The T20 albumin fusion protein of the present invention includes heterologous signal sequences (MAF, INV, Ig, fibrin B, clusterin, insulin-like growth factor binding protein 4, HSA leader sequence variants (chimeric HSA / MAF leader sequences) But is not limited thereto) or other heterologous signal sequences known in the art, including but not limited to. In a preferred embodiment, the T20 albumin fusion protein of the invention comprises the native HIV-1 transmembrane protein gp41 signal sequence. In a further preferred embodiment, the T20 albumin fusion protein of the invention further comprises an N-terminal methionine residue. Polynucleotides encoding these polypeptides (including fragments and / or variants) are also encompassed by the present invention.

(Expression and purification of construct ID 2673)
(Expression in yeast S. cerevisiae)
Construct 2673 is obtained from yeast S. coli by methods known in the art. cerevisiae can be transformed (see Example 3). Expression levels can be tested by immunoblot detection using anti-HSA serum as the primary antibody.

(Purification from the supernatant of yeast S. cerevisiae cells)
Yeast S. In cerevisiae, the cell supernatant containing the secreted T20 albumin fusion protein expressed from construct ID number 2673 can be purified as described in Example 4. N-terminal sequencing of the expressed and purified albumin fusion protein should yield YTSLI (SEQ ID NO: 2151) corresponding to the amino terminus of HIV-1 inhibitory peptide T20.

(The activity of the T20 albumin fusion encoded by Construct ID No. 2673 can be assayed using in vitro infectivity assays and / or cell-cell fusion inhibition assays)
(Method)
The T20 albumin fusion protein encoded by construct 2673 is described in Example 108 under the subsection title “T20 activity can be assayed using in vitro infectivity assay and / or cell-cell fusion inhibition assay”. As described above, it can be tested in in vitro infectivity bioassays and cell-cell fusion inhibition assays.

Example 110: Indicator for T20 albumin fusion protein
Based on the activity of the T20 albumin fusion protein in the above assay, the T20 albumin fusion protein is useful in the treatment, prevention, and / or diagnosis of HIV, AIDS and / or SIV (simian immunodeficiency virus) infection.

(Example 111: Generation of construct ID 2667 (HSA-T1249))
Construct ID 2667 (pSAC35: HSA.T1249) is designated as yeast S. cerevisiae. cerevisiae expression vector pSAC35 encodes a T1249 albumin fusion protein having a full-length HSA protein (including native HSA leader sequence) fused to the amino terminus of the second generation fusion inhibitor peptide “T1249” (ie, W1-F39) Containing DNA. This T1249 peptide is a second generation fusion inhibitor derived from the HIV-1 transmembrane protein gp41 and is shown to have inhibitory activity against HIV-1 infection.

(Cloning of T1249 cDNA)
A polynucleotide encoding T1249 was PCR generated using the four overlapping primers described below, T1249-1, T1249-2, T1249-3 and T1249-4. This sequence is the yeast S. cerevisiae. Codon optimized for expression in C. cerevisiae. The PCR fragment was cut with Bsu 36I / Asc I and ligated into pScNHSA cut with Bsu 36I / Asc I. The Not I fragment was then subcloned into the pSAC35 plasmid. Construct ID number 2667 encodes an albumin fusion protein comprising the full length HSA protein (including the native HSA leader sequence) fused to the T1249 peptide (ie, Trp-1 to Phe-39).

  Four overlapping oligonucleotide 5 'and 3' primers (T1249-1 and T1249-4) suitable for PCR amplification of the polynucleotide encoding the T1249 peptide were synthesized.

Ｔ１２４９−１は、Ｂｓｕ ３６Ｉクローニング部位（下線で示される）およびＨＳＡの成熟形態（配列番号１０３８）の最後の４アミノ酸残基をコードするヌクレオチドおよびＴ１２４９ペプチドの最初の７アミノ酸残基（すなわち、Ｔｒｐ−１〜Ｌｙｓ−７）をコードする２１ヌクレオチド（太字で示される）を組み込む。Ｔ１２４９−４において、Ａｓｃ Ｉ部位は下線が引かれ、そして最後の３０ヌクレオチド（太字で示される）は、Ｔ１２４９ペプチドの最後の１０アミノ酸残基（Ａｓｐ−３０〜Ｐｈｅ−３９）をコードするＤＮＡの逆相補体である。Ｔ１２４９−２オリゴヌクレオチドおよびＴ１２４９−３オリゴヌクレオチドは、互いに、そしてそれぞれＴ１２４９−１およびＴ１２４９−４と重複し、そしてＴ１２４９ペプチドをコードする。ＰＣＲ産物を精製し（例えば、Ｗｉｚａｒｄ ＰＣＲ Ｐｒｅｐｓ ＤＮＡ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｓｙｓｔｅｍ（Ｐｒｏｍｅｇａ Ｃｏｒｐ）を使用して）、そしてＢｓｕ ３６ＩおよびＡｓｃ Ｉで消化した。ゲル電気泳動によるＢｓｕ ３６Ｉ−Ａｓｃ Ｉフラグメントのさらなる精製後、この産物を、Ｂｓｕ ３６Ｉ／Ａｓｃ Ｉで消化したｐＳｃＮＨＳＡにクローニングした。配列を確認した後、このＴ１２４９アルブミン融合タンパク質をコードする発現カセットを、Ｎｏｔ ＩフラグメントとしてｐＳＡＣ３５にサブクローニングした。Ｎｏｔ Ｉフラグメントをさらに、ｐＳＡＣ３５にサブクローニングして、構築ＩＤ番号２６６７を供与した。

T1249-1 is the nucleotide encoding the Bsu 36I cloning site (shown underlined) and the last 4 amino acid residues of the mature form of HSA (SEQ ID NO: 1038) and the first 7 amino acid residues of the T1249 peptide (ie Trp Incorporates 21 nucleotides (shown in bold) encoding -1 to Lys-7). In T1249-4, the Asc I site is underlined and the last 30 nucleotides (shown in bold) are those of the DNA encoding the last 10 amino acid residues (Asp-30-Phe-39) of the T1249 peptide. Reverse complement. The T1249-2 and T1249-3 oligonucleotides overlap each other and T1249-1 and T1249-4, respectively, and encode the T1249 peptide. The PCR product was purified (eg, using the Wizard PCR Preps DNA Purification System (Promega Corp)) and digested with Bsu 36I and Asc I. After further purification of the Bsu 36I-Asc I fragment by gel electrophoresis, this product was cloned into pScNHSA digested with Bsu 36I / Asc I. After confirming the sequence, the expression cassette encoding this T1249 albumin fusion protein was subcloned into pSAC35 as a Not I fragment. The Not I fragment was further subcloned into pSAC35 and provided construction ID number 2667.

  Furthermore, N-terminal analysis of the expressed albumin fusion protein by amino acid sequencing can confirm the presence of the expressed HSA sequence (see below).

  The T1249 albumin fusion protein of the present invention is preferably a mature form of HSA (ie, fused to either the N-terminus or C-terminus of HIV-1 inhibitory peptide T1249 (ie, Trp-1 to Phe-39)) Asp-25 to Leu-609). In one embodiment of the invention, the T1249 albumin fusion protein of the invention further comprises a signal sequence that directs the nascent fusion polypeptide in the host secretory pathway used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed and the mature T1249 albumin fusion protein is secreted directly into the culture medium. The T1249 albumin fusion protein of the present invention includes heterologous signal sequences (MAF, IFV, Ig, fibrin B, clusterin, insulin-like growth factor binding protein 4, HSA leader sequence variants (including chimeric HSA / MAF leader sequences, And other heterologous signal sequences known in the art, including but not limited to). In a preferred embodiment, the T1249 albumin fusion protein of the invention comprises the native HIV-1 transmembrane protein gp41 signal sequence. In a further preferred embodiment, the T1249 albumin fusion protein of the invention further comprises an N-terminal methionine residue. Polynucleotides encoding these polypeptides (including fragments and / or variants) are also encompassed by the present invention.

(Expression and purification of construct ID 2667)
(Expression in yeast S. cerevisiae)
Construct 2667 is obtained from yeast S. cerevisiae by methods known in the art (see Example 3). can be transformed into cerevisiae. Expression levels can be tested by immunoblot detection using anti-HSA serum as the primary antibody.

(Purification from the supernatant of yeast S. cerevisiae cells)
Cell supernatants containing secreted T1249 albumin fusion protein (expressed from construct ID # 2667 in yeast S. cerevisiae) can be purified as described in Example 4. N-terminal sequencing of the albumin fusion protein yields the sequence DAHKS (corresponding to the amino terminus of the mature form of HSA).

(The activity of the T1249 albumin fusion protein encoded by Construct ID # 2667 can be assayed using in vitro infectivity assays and / or cell-cell fusion inhibition assays)
(Method)
The T1249 albumin fusion protein encoded by construct 2667 is described above under the heading “The activity of T20 can be assayed using an in vitro infectivity assay and / or an intercellular fusion inhibition assay” in Example 108. As such, it can be tested in in vitro infectivity bioassays and cell-cell fusion inhibition assays.

(Example 112: Construction ID 2670 (T1249-HSA) generation)
Construct ID 2670, pSAC35: T1249. HSA contains DNA encoding a T1249 albumin fusion protein. This fusion protein is an HSA chimeric leader sequence (ie, HSA-kex2 signal peptide, second generation fusion inhibitor peptide, “T1249” (ie, W1 fused to the amino terminus of the mature form of HSA in the yeast S. cerevisiae expression vector pSAC35). -F39)).

(Cloning of T1249 cDNA)
DNA encoding the second generation fusion inhibitor peptide was PCR generated using 4 overlapping primers. This sequence is the yeast S. cerevisiae. Codon optimized for expression in C. cerevisiae. The PCR fragment was cut with SalI / ClaI and subcloned into XhoI / ClaI cut pScCHSA. The NotI fragment was then subcloned into the pSAC35 plasmid. Construct ID # 2670 encodes the chimeric leader sequence of HSA fused to the T1249 peptide (ie, Trp-1 to Phe-39), followed by the mature form of HSA.

  Four overlapping oligonucleotide 5 'and 3' primers suitable for PCR amplification of polynucleotides encoding T1249 peptides (T1249-5 and T1249-6) were synthesized:

Ｔ１２４９−５は、ＳａｌＩクローニング部位（下線で示す）、ＨＳＡキメラリーダー配列の最後の３アミノ酸残基をコードするヌクレオチド、およびＴ１２４９ペプチドの最初の７アミノ酸（すなわち、Ｔｒｐ−１〜Ｌｙｓ−７）をコードするＤＮＡ（太字で示す）を、組み込んでいる。Ｔ１２４９−６において、下線で示される配列は、ＣｌａＩ部位である；そしてＣｌａＩ部位およびそれに続くＤＮＡは、成熟ＨＳＡタンパク質（配列番号１０３８）の最初の１０アミノ酸をコードするＤＮＡの逆相補鎖である。太字で示される配列は、Ｔ１２４９ペプチドの最後の１０アミノ酸残基（Ａｓｐ−３０〜Ｐｈｅ−３９）をコードする、３０ヌクレオチドの逆相補鎖である。Ｔ１２４９−２オリゴヌクレオチドおよびＴ１２４９−３オリゴヌクレオチドは、（実施例１１１のように）互いに重複し、そしてそれぞれＴ１２４９−５オリゴヌクレオチドおよびＴ１２４９−６オリゴヌクレオチドに重複し、そしてＴ１２４９ペプチドをコードする。これらのプライマーを使用し、アニーリングさせ、アニーリングさせたプライマーを伸長させ、ＳａｌＩおよびＣｌａＩを用いて切断し、そしてＸｈｏＩ／ＣｌａＩ切断したｐＳｃＣＨＳＡ内にサブクローニングすることによって、Ｔ１２４９ペプチドを、産生した。配列を確認した後、Ｔ１２４９アルブミン融合発現カセットを含むＮｏｔＩフラグメントを、ＮｏｔＩを用いて切断したｐＳＡＣ３５内にサブクローニングし、構築物ＩＤ２６７０を生成させた。構築物ＩＤ＃２６７０は、キメラリーダー配列、Ｔ１２４９ペプチド、およびＨＳＡの成熟形態を含むアルブミン融合タンパク質を、コードする。

T1249-5 contains a SalI cloning site (shown underlined), the nucleotide encoding the last 3 amino acid residues of the HSA chimeric leader sequence, and the first 7 amino acids of the T1249 peptide (ie, Trp-1 to Lys-7). Encoding DNA (shown in bold) is incorporated. In T1249-6, the underlined sequence is a ClaI site; and the ClaI site and the DNA following it are the reverse complement of DNA encoding the first 10 amino acids of the mature HSA protein (SEQ ID NO: 1038). The sequence shown in bold is the 30 nucleotide reverse complement that encodes the last 10 amino acid residues (Asp-30-Phe-39) of the T1249 peptide. The T1249-2 and T1249-3 oligonucleotides overlap each other (as in Example 111) and overlap the T1249-5 and T1249-6 oligonucleotides, respectively, and encode the T1249 peptide. The T1249 peptide was produced by using these primers, annealing, extending the annealed primer, cleaving with SalI and ClaI, and subcloning into XhoI / ClaI cleaved pScCHSA. After confirming the sequence, the NotI fragment containing the T1249 albumin fusion expression cassette was subcloned into pSAC35 cut with NotI to generate construct ID2670. Construct ID # 2670 encodes an albumin fusion protein comprising a chimeric leader sequence, a T1249 peptide, and a mature form of HSA.

  Furthermore, analysis of the N-terminus of the expressed albumin fusion protein by amino acid sequencing can confirm the presence of the expected T1249 sequence (see below).

  The T1249 albumin fusion protein of the present invention preferably comprises a mature form of HSA (ie, Asp-25-Leu-609), which is a T1249 peptide (ie, Trp-1 to Phe-39). To either the N-terminus or the C-terminus. In one embodiment of the invention, the T1249 albumin fusion protein of the invention further comprises a signal sequence that leads to the host secretory pathway used for expression of the newly produced fusion polypeptide. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed and the mature T1249 albumin fusion protein is secreted directly into the culture medium. The T1249 albumin fusion protein of the invention can include a heterologous signal sequence. Heterologous signal sequences include, but are not limited to, MAF, INV, Ig, fibrin B, clusterin, insulin-like growth factor binding protein 4, variant HSA leader sequence. Variant HSA leader sequences include, but are not limited to, chimeric HSA / MAF leader sequences, or other heterologous signal sequences known in the art. In a preferred embodiment, the T1249 albumin fusion protein of the invention comprises the native HIV-1 transmembrane protein gp41 signal sequence. In a further preferred embodiment, the T1249 albumin fusion protein of the present invention further comprises an N-terminal methionine residue. Polynucleotides (including fragments and / or variants) encoding these polypeptides are also included in the present invention.

(The activity of the T1249 albumin fusion encoded by Construct ID # 2670 can be assayed using an in vitro infectivity assay and / or a cell-cell fusion inhibition assay)
(Method)
The T1249 albumin fusion protein encoded by construct 2670 is described in the subheading “Activity of T20 can be assayed using in vitro infectivity assay and / or cell-cell fusion inhibition assay” in Example 108 above. As such, it can be tested in in vitro infectivity bioassays and cell-cell fusion inhibition assays.

(Example 113: Adaptation of T1249 albumin fusion protein)
Based on the activity of the T1249 albumin fusion protein in the above assay, the T1249 albumin fusion protein is useful in the treatment, prevention, and / or diagnosis of HIV, AIDS, and / or SIV (simian immunodeficiency virus) infection.

  (Example 114: Construction ID 2702 (HSA-GCSF.T31-L201) production) Construction ID 2702, pSAC35: HSA. GCSF. T31-L201 contains DNA encoding GCSF albumin fusion protein. This construct is the yeast S. cerevisiae. The cerevisiae expression vector pSAC35 has mature HSA fused downstream of the HSA / kex2 leader sequence and upstream of the GCSF T31-L201 amino acids.

(GCSF cDNA cloning)
The polynucleotide encoding the GCSF C-terminal deletion mutant was PCR amplified using the GCSF-5 and GCSF-6 primers described below. This amplification product was cut with Bsu36I / AscI and subcloned into pScNHSA. Construct ID # 2702 encodes an albumin fusion protein comprising mature HSA fused downstream of the HSA / kex2 leader sequence and upstream of amino acids T31-L201 of GCSF.

  Two oligonucleotide (GCSF-5 and GCSF-6) primers suitable for PCR amplification of polynucleotides encoding the GCSF C-terminal deletion mutant were synthesized:

ＧＣＳＦ−５は、Ｂｓｕ３６Ｉクローニング部位（下線で示す）、ならびにＨＳＡの最後の６アミノ酸、および成熟ＧＣＳＦの最初の６アミノ酸（アミノ酸Ｔ３１〜Ａ３６）をコードするヌクレオチドを、組み込んでいる。ＧＣＳＦ−６は、ＡｓｃＩクローニング部位（下線で示す）を含み、そして最後の２５塩基は、ＧＣＳＦ Ｃ末端欠失変異体の最後の８アミノ酸残基（Ｓ１９４〜Ｌ２０１）をコードするＤＮＡの逆相補鎖である。これらのプライマーを用いて産生されたＰＣＲ産物を精製し（例えば、Ｗｉｚａｒｄ ＰＣＲ Ｐｒｅｐｓ ＤＮＡ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｓｙｓｔｅｍ（Ｐｒｏｍｅｇａ Ｃｏｒｐｏｒａｔｉｏｎ）を使用して）、次いでＢｓｕ３６ＩおよびＡｓｃＩを用いて切断した。ゲル電気泳動法によるＢｓｕ３６Ｉ／ＡｓｃＩ ＰＣＲフラグメントのさらなる精製の後、この産物を、Ｂｓｕ３６Ｉ／ＡｓｃＩ切断されたｐＳｃＮＨＳＡ内にクローン化した。配列を確認した後、このＧＣＳＦアルブミン融合タンパク質をコードする発現カセットを、ｐＳＡＣ３５内に、ＮｏｔＩフラグメントとしてサブクローニングした。

GCSF-5 incorporates a Bsu36I cloning site (shown underlined), as well as nucleotides encoding the last 6 amino acids of HSA and the first 6 amino acids of mature GCSF (amino acids T31-A36). GCSF-6 contains an AscI cloning site (underlined) and the last 25 bases are the reverse complementary strand of DNA encoding the last 8 amino acid residues (S194-L201) of the GCSF C-terminal deletion mutant It is. The PCR product produced using these primers was purified (eg, using the Wizard PCR Preps DNA Purification System (Promega Corporation)) and then cleaved using Bsu36I and AscI. After further purification of the Bsu36I / AscI PCR fragment by gel electrophoresis, the product was cloned into Bsu36I / AscI cut pScNHSA. After confirming the sequence, the expression cassette encoding this GCSF albumin fusion protein was subcloned into pSAC35 as a NotI fragment.

  Further analysis by amino acid sequencing of the N-terminus of the expressed albumin fusion protein can confirm the presence of the expected HSA sequence (see below).

  The GCSF albumin fusion protein of the present invention preferably comprises a mature form of HSA (ie Asp-25 to Leu-609), which is a GCSF C-terminal deletion mutant (ie T31 to L201). It is fused to either the N-terminus or C-terminus. In one embodiment of the invention, the GCSF albumin fusion protein of the invention further comprises a signal sequence that directs the newly produced fusion polypeptide into the secretory pathway of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed and the mature GCSF albumin fusion protein is secreted directly into the culture medium. The GCSF albumin fusion protein of the invention can include a heterologous signal sequence. Examples of heterologous signal sequences include MFα-1, invertase, Ig, fibrin B, clusterin, insulin-like growth factor binding protein 4, K.I. lactis killing toxin (K. lactis killer toxin), and variant HSA leader sequences. Variant HSA leader sequences include chimeric HSA / MFα-1 (HSA / kex2) leader sequences; lactis / MFα-1 leader sequences, chimeras or other heterologous signal sequences known in the art, including but not limited to. In a further preferred embodiment, the GCSF albumin fusion protein of the invention comprises a native GCSF signal sequence. In a further preferred embodiment, the GCSF albumin fusion protein of the present invention further comprises an N-terminal methionine residue. Polynucleotides (including fragments and / or variants) encoding these polypeptides are also included in the present invention.

(Expression and purification of construct ID # 2702)
(Expression in yeast S. cerevisiae)
Construct # 2702 was prepared from yeast S. cerevisiae by methods known in the art (see Example 3) and as described above for construct ID # 1642 (see Example 19). transformed into cerevisiae. Expression levels were tested by immunoblot detection using anti-HSA serum as the primary antibody (data not shown).

(Purification from the supernatant of yeast S. cerevisiae cells)
The general procedure for the purification of albumin fusion protein is described in Example 4. Yeast S. The cell supernatant containing the GCSF albumin fusion protein expressed from construct ID # 2702 in cerevisiae was purified as described in Example 20. N-terminal sequencing of the albumin fusion protein yields the sequence DAHKS (corresponding to the amino terminus of the mature form of HSA).

(The activity of the GCSF albumin fusion protein encoded by construct # 2702 can be assayed using an in vitro NFS-60 cell proliferation assay)
(Method)
The GCSF albumin fusion protein encoded by construct 2702 is encoded by the subheadings “GCSF activity can be assayed using an in vitro NFS-60 cell proliferation assay” in Example 19 and “construct ID # 1642”. The activity of the GCSF albumin fusion protein could be assayed using an in vitro NFS-60 cell proliferation bioassay, as described above under “Can be assayed using an in vitro NFS-60 cell proliferation assay”.

(result)
Both the partially purified GCSF albumin fusion protein encoded by construct 1634 (HSA-GCSF) and the GCSF C-terminal deletion mutant albumin fusion protein (L-171) encoded by construct 2702 are NFS- Demonstrating the ability to cause 60 cell proliferation, the C-terminal deletion mutant showed a stronger proliferation effect (see FIG. 19). Unexpectedly, the fusion protein encoded by construct 2702 showed 2-3 times higher activity than the fusion protein encoded by construct 1643. Alternative GCSF albumin fusion constructs include albumin fused to amino acid residues 1-169 of mature GCSF and albumin fused to amino acid residues 1-170 of mature GCSF.

(Example 115: Construct ID 2876 (HSA-IFNα hybrid))
Construct ID 2876 pSAC35: HSA. IFNαA (C1-Q91) / D (L93-E166) R23K, A113V contains DNA encoding IFNα hybrid albumin fusion protein, which is cerevisiae expression vector pSAC35 has mature HSA fused downstream of the HSA / kex2 leader sequence and upstream of the amino acid sequence of the IFNαA / D hybrid. With regard to the composition of hybrid IFN, the first 91 amino acids are derived from IFNα2 (also called IFNαA) subtype and the remaining 75 amino acids are derived from IFNα1 (IFNαD). We have incorporated two point mutations (R23K, A113V). This fusion was produced by PCR and fused downstream of HSA in the yeast expression vector pSAC35.

(result)
(CID 2876 expression and purification)
Yeast strain BXP-10 was transformed with pSAC35: CID2876 and transformants were selected for fermentation. A 5 liter fermentation was performed and the supernatant was analyzed, demonstrating high expression (approximately 500 mg / l). A small portion of the supernatant was experimentally purified. Approximately 1 mg of CID2876 protein (95% or more pure based on N-terminal sequence) was obtained after purification through blue sepharose, followed by gel filtration and then Q-anion conversion. The remaining fermentation starting material is available for further purification as required.

(ISRE activity)
All type I IFNs mediate their activity through the involvement of a common IFN receptor complex and activation of the ISRE signaling pathway. Activation of gene transcription through this pathway results in cellular responses associated with IFN, including antiproliferation, antiviral and immunomodulation. Using a reporter-based strategy, the ability of CID 2876 to activate the ISRE signaling pathway was determined. CID 2876 was found to be a potent activator of the ISRE pathway and demonstrated an EC _{50 of} 2.7 ng / ml (data not shown). This result is comparable to the potency of CID 3165 in this assay system.

(Antiviral activity)
An important activity of IFN is its ability to mediate cytoprotection against viral infection of IFN. Most human type I IFNs show antiviral activity in a species-limited manner, while the hybrid IFN used in this study has been demonstrated to be active in mouse cells. Therefore, the antiviral activity of CID 2876 was evaluated in the mouse cell line L929 infected by EMCV. The results show that CID 2876 demonstrates antiviral activity in a cross-species manner (data not shown).

Example 116: Activity of construct 3070 (GLP-1 albumin fusion) as measured by in vitro stimulation of insulin mRNA in INS-1 cells
Recently, GLP-1 has been shown to increase insulin mRNA expression in pancreatic β cells (Buteau et al., Diabetologia 1999 Jul; 42 (7): 856-64). Therefore, the ability of the GLP-1 albumin fusion protein encoded by CID 3070 to stimulate insulin mRNA was evaluated using the pancreatic β cell line INS-1 (832/13).

  FIG. 14 shows the steady state level of insulin mRNA after treatment with GLP-1 or GLP-1 albumin fusion protein encoded by construct ID3070 (CID 3070 protein) in INS-1 (832/13) cells. , Illustrated. Both GLP-1 and CID 3070 proteins stimulate transcription of the insulin gene. The first bar (black) represents untreated cells. Bars 2-4 (white) represent cells treated with the indicated concentrations of GLP-1. Bars 5-7 (grey) represent cells treated with the indicated concentrations of CID 3070 protein.

  It will be apparent that the invention may be practiced otherwise than as particularly described in the foregoing description and examples. Many modifications and variations of this invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims.

  The entire disclosure of each cited document (patents, patent applications, patent publications, journal articles, abstracts, laboratory manuals, books, or other disclosures) is referenced in the database (GenBank, GeneSeq, As well as information obtained through an identifier specific to CAS Registry, etc.) is hereby incorporated by reference in its entirety.

  In addition, the specification and sequence listing of each of the following US applications is incorporated herein by reference in its entirety: US Application No. 60 / 341,811 (filed December 21, 2001); US Application No. 60 / 360,000 (filed February 28, 2002); US Application No. 60 / 378,950 (filed May 10, 2002); US Application No. 60 / 398,008 (Filed July 24, 2002); U.S. Application No. 60 / 411,355 (filed Sep. 18, 2002); U.S. Application No. 60 / 414,984 (filed Oct. 2, 2002); US Application No. 60 / 417,611 (filed October 11, 2002); US Application No. 60 / 420,246 (filed October 23, 2002); US Application Number 60 / 423,62 (Filed on Nov. 5, 2002); U.S. Application No. 60 / 350,358 (filed on Jan. 24, 2002); U.S. Application No. 60 / 359,370 (filed on Feb. 26, 2002) U.S. Application No. 60 / 367,500 (filed March 27, 2002); U.S. Application No. 60 / 402,131 (filed Aug. 9, 2002); U.S. Application No. 60 / 402,708 No. (filed Aug. 13, 2002); U.S. Application No. 60 / 351,360 (filed Jan. 28, 2002); U.S. Application No. 60 / 382,617 (filed May 24, 2002) U.S. Application No. 60 / 383,123 (filed May 28, 2002); U.S. Application No. 60 / 385,708 (filed Jun. 5, 2002); U.S. Application No. 60 / 394,625 No. (20 U.S. Application No. 60 / 411,426 (filed Sep. 18, 2002); U.S. Application No. 60 / 370,227 (filed Apr. 8, 2002); Application No. 60 / 351,360 (filed Jan. 28, 2002); U.S. Application No. 60 / 382,617 (filed May 24, 2002); U.S. Application No. 60 / 383,123 ( Filed May 28, 2002); U.S. Application No. 60 / 385,708 (filed Jun. 5, 2002); U.S. Application No. 60 / 394,625 (filed July 10, 2002); and U.S. Application No. 60 / 411,426 (filed Sep. 18, 2002). Further, concurrently with this specification, Human Genome Sciences, Inc., filed on December 23, 2002. The specification and sequence listing of the US application with agent docket number PF574PCT is hereby incorporated by reference in its entirety.

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(Canada)
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(Norway)
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(Australia)
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(Finland)
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(UK)
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(Sweden)
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Claims (12)

An albumin fusion protein comprising an insulin polypeptide fused to albumin, said fusion protein having insulin activity;
(A) a full-length human albumin polypeptide fused to the amino terminus of a synthetic single chain insulin analog encoded by construct ID 2250, wherein the synthetic single chain insulin analog is an insulin growth factor (instead of a proinsulin C-peptide) IGF-1) having a C domain,
(B) A chimeric human serum albumin (HSA) leader sequence fused to the amino terminus of a synthetic single chain insulin analog encoded by construct ID 2255, wherein the synthetic single chain insulin analog is insulin instead of proinsulin C-peptide Having a growth factor (IGF-1) C domain and the synthetic single chain insulin analogue is fused to the amino terminus of a mature human albumin polypeptide;
(C) Full length human albumin polypeptide fused to the amino terminus of a synthetic single chain insulin analog encoded by construct ID 2276, the synthetic single chain insulin analog has a synthetic linker instead of proinsulin C-peptide Or
(D) a chimeric HSA leader sequence encoded by construct ID 2278, fused to the amino terminus of a synthetic single chain insulin analog, the synthetic single chain insulin analog has a synthetic linker instead of a proinsulin C-peptide The synthetic single chain insulin analogue is fused to the amino terminus of a mature human albumin polypeptide;
An albumin fusion protein comprising: A construct encoding the albumin fusion protein of claim 1 . ID2250,2255,2276, and 2278 or we selected the claims 2 constructs described. The construct of claim 2 , comprising a nucleotide sequence of ATCC accession number PTA-3916, PTA-3913, or PTA-3918, wherein the nucleotide sequence encodes an insulin polypeptide sequence fused to albumin. A host cell that expresses the albumin fusion protein according to claim 1 . 6. The host cell according to claim 5 , which is a mammalian cell, yeast cell or prokaryotic cell. A method for expressing an albumin fusion protein, comprising culturing the host cell according to claim 5 under conditions suitable for the expression of an albumin fusion protein, and recovering the albumin fusion protein. 6. An albumin fusion protein expressed by a host cell according to claim 5 , which is glycosylated, non-glycosylated or a glycosylated isomer. A composition comprising the albumin fusion protein of claim 1 and a pharmaceutically acceptable carrier. A kit comprising the composition of claim 9 and instructions for its use. A pharmaceutical composition for treating, preventing, diagnosing or ameliorating a disease, disorder or condition comprising an effective amount of the albumin fusion protein according to claim 1 . Disease, disorder, or condition is hyperglycemia, diabetes, diabetes insipidus, diabetes mellitus, type 1 diabetes, type 2 diabetes, insulin resistance, insulin deficiency, hyperlipidemia, hyperketonuria, non-insulin dependent intrinsic Diabetes, insulin-dependent diabetes mellitus, metabolic disorders, immune disorders, obesity, tube disorders, weight suppression, appetite suppression, X syndrome, diabetes-related obesity, diabetes-related heart disease, diabetes-related hyperglycemia, 12. A pharmaceutical composition according to claim 11 , selected from diabetes-related infections, diabetes-related retinopathy, and diabetes-related ulcers.

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